Electrochemical behavior of a novel nano-composite coat on Ti alloy in phosphate buffer solution for biomedical applications

Abstract

A novel nano-composite film coat of organic/inorganic composition including chitosan (CS), TiO₂ nanoparticles (TO) and hydroxyapatite (HA) nanoparticles, was synthesized on a Ti–6Al–4V alloy surface. Open-circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization measurements were used to observe the corrosion behavior of the novel synthesized nano-composite coat on titanium alloy surface in a phosphate buffer solution. The results were confirmed by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis techniques. The antibacterial activity for the novel nano-coat was determined and compared with the bare Ti alloy. Electrochemical impedance spectroscopy measurements showed that the total corrosion resistance of this newly synthesized nano-coat gave the highest corrosion resistance compared to the separate CS, TO and/or HA coatings. Also, this novel nano-coat showed high antibacterial activity compared with the bare alloy. The excellent biocompatibility of this novel nano-coat may be due to its organic/inorganic composition.

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1. Introduction

Titanium alloys have been used as implants in the human body¹ and are essential metallic biomaterials that can replace hard tissue in orthopedic and dental applications. This applicability is attributed to its high corrosion resistance, biocompatibility, low Young's modulus² and low specific weight.³ A Ti alloy was the first registered material in the ASTM standards (F-136-84)^4,5 as an implant that is used mostly for dental, orthopedic and osteosynthesis applications.^6–10

Film resistance plays an important role in determining the successful use of metal alloys as biomaterials. The development of a non-toxic and effective coverage film on the Ti alloy is an important challenge to improve its stability and resistance, which is necessary for many applications. Chitosan is recommended as a suitable functional material, because of its excellent biocompatibility, biodegradability, nontoxicity and adsorption properties. It is an alkaline-deacetylated chitin derived from the exoskeletons of insects and shells of crustaceans.^10–13 Hydroxyapatite [HA, Ca₁₀(PO₄)₆(OH)₂] has been used as a coat for a Ti alloy surface to improve both its implant-tissue osseointegration and biocompatibility.² It can be used as a coat in the field of orthopedics and dentistry owing to its chemical and biological similarity to human hard tissues. It shows a good fixation to the host bone and increased bone ingrowth to the implant. A TiO₂ nanoparticle coating has been used to improve the corrosion resistance of Ti alloys.¹⁵ Phosphate-buffered saline (abbreviated PBS) is a buffer solution commonly used in biological research.

Antibacterial activity has been studied to examine the use of newly synthesized nano-coats on a Ti alloy as a biomedical implant¹⁴ and to enhance its antimicrobial ability and reduce its failure as an implant.

Therefore, this study was performed to formulate a novel nano-composite coat (chitosan/HA/TiO₂) containing an organic/inorganic nano-layer on a Ti alloy surface, in order to optimize its biocompatibility. The coated Ti alloy was studied in phosphate buffer solution (PBS) of pH 7.4 at 37 °C with immersion time via electrochemical impedance spectroscopy (EIS), open circuit potential (OCP) and potentiodynamic polarization techniques. The newly synthesized nano-coat was characterized by both SEM and EDX techniques. Finally, the antibacterial activity of this novel nano-coat was studied and compared to the bare alloy to observe its potential use as an anti-biocorrosion agent.

2. Experimental

2.1. Electrode and cell composition

The Ti–6Al–4V alloy was taken from Johnson and Matthey (England). Its chemical composition (wt%) is 5.7 Al, 3.85 V, 0.18 Fe, 0.038 C, 0.106 O and 0.035 N and balance titanium. Its cross sectional area is 0.196 cm². The alloy was coated with Araldite epoxy resin then welded to an electrical wire and finally fixed in a cylindrical glass tube. Polishing of the alloy was performed using abrasive paper (400–1000 grades), degreased in acetone, washed by ethanol and then dried in air. A conventional three-electrode cell, consisting of the tested alloy (Ti alloy) as a working electrode, a large platinum sheet as a counter electrode (CE) and saturated calomel electrode (SCE) as a reference electrode (RE), was used.

2.2. Chemicals and reagents

Chitosan from crab shells (85% deacetylated), acetic acid (≥99.0%), TiO₂ nanoparticles (<100 nm) and a hydroxyapatite nanoparticles dispersion, 10 wt% in water (<200 nm (BET)), were purchased from Sigma-Aldrich. Phosphate buffer solution (PBS) was prepared using Na₃PO₄, Na₂HPO₄, kH₂PO₄, NaCl and KCl (Analar grade reagents) and triply distilled water. The pH was adjusted to 7.4 using HCl.

2.3. Instrumentation

Potentiodynamic polarization curves were measured at a scan rate of 1 mV s⁻¹. The electrochemical impedance spectroscopy experiments were done at 10 mV excitation ac amplitude in a frequency range of 0.1 Hz to 100 Hz. The used electrochemical workstation was an IM6e Zahner-electrik, GmbH, Kronach, Germany, provided with Thales software for I/E and impedance data analyses.

Scanning electron microscopy (SEM) measurements were done using an SEM Model Quanta 250 FEG (Field Emission Gun) connected to an EDX Unit (Energy Dispersive X-ray Analyses), with an accelerating voltage of 30 kV, magnification of 14× up to 1 000 000 and resolution for Gun.1n (FEI company, Netherlands).

All experiments were carried out and repeated 2–3 times at a temperature of 37 °C and gave reproducible results. All potentials were measured and given with respect to SCE (E = 0.241 V per SHE).

2.4. Coating preparation

The first coat (chitosan) was prepared by mixing chitosan with diluted acetic acid (1% w/w) and stirring for 5 hours until the solution became transparent proving that chitosan had been dissolved completely. After that, the solution was filtered and stored at 4 °C.

Secondly, the CS/TiO₂ coat was prepared by adding TiO₂ nanoparticles to the chitosan solution and stirring for 5 hours.

Thirdly, the CS/TiO₂/HA coat was prepared by adding TiO₂ and HA nanoparticles to the chitosan solution and stirring for 5 hours. At the end, each coat obtained was used to cover the electrode surface and left to dry for 2 h before use and/or characterization.

2.5. Antibacterial activity

The newly synthesized nano-composite film CS/TiO₂/HA and the bare Ti alloy were immersed for 20 days in PBS of pH 7.4 at 37 °C. A common Gram-positive pathogen Staphylococcus aureus (National Collection of Industrial Microorganisms) was used to test the antibacterial activity of the CS/TiO₂/HA nano-composite.

2.6. Culture medium

Luria–Bertani (LB) broth was used to culture Staphylococcus aureus at 37 °C with the following composition (g l⁻¹): 5.0 NaCl, 5.0 yeast extraction and 10.0 tryptone. The initial pH for the culture medium was adjusted to 7.2. The test strain was sub-cultured and incubated for 1 h at 310 K. After incubation, the bacterial culture was swabbed on the previously prepared and solidified LB agar plates. The bacterial suspension was diluted to 1 × 10⁶ cfu mL⁻¹ with PBS buffer solution.

The Ti alloy in the form of disks was placed on the Petri dish, one disc was inoculated with the novel CS/TiO₂/HA nano-composite and the bare Ti alloy disk was used as a control. The bacterial resistance, for the CS/TiO₂/HA nano-coated sample compared to the bare alloy, after culturing for 24 h at 37 °C, was examined for a zone of inhibition on the disc.

3. Results and discussion

3.1. Surface morphology and film characterization

A simple chemical synthesis method to fabricate a novel nano-composite coat containing CS/HA/TiO₂ on a Ti electrode is shown in Scheme 1. This coat exhibits biocompatibility which may be due to organic/inorganic composition.

Scheme 1 Schematic illustration for chemical synthesis of nano-coated Ti alloy with CS, TiO₂ NPs and HA NPs.

Fig. 1A–C show SEM images for the (a) CS,¹⁰ (b) CS/TiO₂, and (c) CS/TiO₂/HA coated Ti alloys, respectively. Fig. 1A shows the Ti alloy coated with a chitosan layer and the surface layer is smooth, uniform and folded.¹⁰ SEM images at higher magnifications are shown for the CS/TiO₂ nanoparticles (Fig. 1B), where the TiO₂ nanoparticles are densely packed together within the chitosan matrix. These images show that a compact layer is formed which stabilizes the Ti alloy surface by forming a protective compact film. Fig. 1C shows the SEM images at two different magnifications of the CS/TiO₂/HA coat. This coat is stacked in an ice shape which improves the alloy corrosion resistance by preventing aggressive ions from PBS forming on the alloy surface.

Fig. 1 SEM images of (A) CS,¹⁰ (B) CS/TiO₂, and (C) CS/TiO₂/HA nano-coated Ti alloys.

Fig. 2A and B show the EDX analysis of the CS/TiO₂ and CS/TiO₂/HA nano-coats, respectively. The elemental composition of CS/TiO₂ was investigated (Fig. 2A) and confirms the presence of a significant amount of elemental carbon (5.22%), oxygen (24.44%) and titanium (66.36%), the main elements of chitosan and TiO₂ nanoparticles. EDX analysis (Fig. 2C) of the CS/TiO₂/HA nano-composite coat confirms the presence of C (4.55%), O (41.12%), Ti (32.21%) and Ca (0.67%) which ensures the presence of HA in the coat. EDX analysis shows a decrease in Ti percentage or an increase in oxygen percentage in the CS/TiO₂/HA nano-composite which implies good coverage of HA nanoparticles on the TiO₂ nanoparticle and chitosan coat. TiO₂ nanoparticles were used to increase the strength of HA to the Ti alloy surface in the chitosan matrix¹⁵ and the chitosan matrix was used to increase the adsorption of both TiO₂ and HA nanoparticles to the Ti alloy surface.^10–13

Fig. 2 EDX analysis of (A) the CS/TiO₂ and (B) CS/TiO₂/HA nano-coated Ti alloys.

3.2. Open circuit potential and impedance measurements

The open circuit potentials (OCPs) of the bare Ti alloy, CS, CS/TiO₂ and CS/TiO₂/HA coats were studied after immersion in PBS at pH 7.4 and 37 °C for 20 days. Fig. 3 shows the variation in steady state open circuit potential (E) with time for the four tested samples. Studying OCP is a way to investigate the corrosion behavior of the studied electrodes and it indicates a tendency of the tested electrode towards electrochemical dissolution/passivation in a test solution. The E value shifts to less negative values with increasing immersion time indicating film healing and thickening for all studied coats. Also, the OCP value shifts negatively in the following order: CS/TiO₂/HA (−120 mV) > CS/TiO₂ (−188 mV) > CS (−304 mV) > bare Ti alloy (−348 mV).

Fig. 3 Variation with time of steady state potential (E) for the four electrodes in PBS at 37 °C.

This indicates that adding HA nanoparticles and TiO₂ nanoparticles into the chitosan matrix improves the passivation of the Ti alloy as an implant by forming a strong passive film on the alloy surface indicated by a stable and lowest negative OCP (−120 mV). So, this study was performed to synthesize a nano-coat of CS/TiO₂/HA on a Ti alloy surface to optimize its biocompatibility as an implant.

Titanium alloy has been used before as an implant material to improve the osseointegration of an implant-tissue.¹⁶ Chitosan is an important functional material, because of its exceptional high adsorption properties and biocompatibility.¹⁷ Thus it can adsorb well onto a Ti alloy surface protecting it from corrosion by shifting its OCP to −304 mV which is less negative than that for the bare electrode (−348 mV). The addition of TiO₂ nanoparticles into the chitosan matrix shifts the OCP to a less negative potential confirming higher protection for the Ti alloy surface. This is due to the high strength property of titanium oxide nanoparticles that increases the film strength on the alloy surface.¹⁵ Modification of the Ti alloy surface is finally performed by adding hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) nanoparticles that provide good fixation to the bone with improved ingrowth for the bone. This improvement in its biocompatibility is due to the chemical and biological similarity of HA to hard tissues, and its consequent direct bonding to host bones.²

Generally, the results indicate that the CS/TiO₂/HA nano-coat is the most protective coat with the lowest negative E value and the best performance out of all the coats.

Impedance measurements can give information about the electrochemical behavior of both the uncoated and nano-coated electrodes. Fig. 4a and b show Bode plots of the bare Ti electrode after immersion in PBS for 20 days, at 37 °C. They show different shapes in the phase diagrams with a phase angle maximum near to 80°. The impedance values increase sharply with increasing time till 5 days then they become stable till 20 days. The same happens for the phase angle maximum which increases from nearly 66° to near 81°. This means that the formed oxide film is stable for 20 days.

Fig. 4 Bode plots of bare Ti–6Al–4V alloy in PBS with immersion time (a) 0–24 h; (b) 2–20 days, at 37 °C.

The best model that fits the experimental data is shown in Fig. 5. It is a three-time constant equivalent circuit model.¹⁰ R_s is the solution resistance and R₁ and R₂ are the resistances of the two formed layers. R₁ is the innermost layer and R₂ is the outermost layer. W is the Warburg impedance due to the diffusion process. Three parallel constant-phase elements (C₁, C₂ and C₃) are shown in the model. A constant-phase element (CPE) was used instead of the ideal capacitance to account for surface heterogeneity.¹⁸ The impedance of the CPE is Z_CPE = [C(jw)^α]⁻¹, where −1 ≤ α ≤ 1. The α value is due to the surface inhomogeneity. The resistance and capacitance values for the bare alloy are given in Table 1.

Fig. 5 Three time constant equivalent circuit including Warburg impedance.

Table 1 Fitting parameters of the test alloy in PBS with immersion time, at 37 °C

Electrodes	Time/h	R 1/MΩ cm^2	CPE1/µF cm^−2	R 2/Ω cm^2	CPE2/µF cm^−2	R 3/KΩ cm^2	CPE3/µF cm^−2	W/kΩ cm^2 s^−1/2	R s/Ω
Ti alloy	0.0	1.3	2.3	11	5.3	4.8	40.8	1.8	19.9
	0.5	1.6	2.1	18	5.4	6.6	36.6	2.2	22.9
	3.0	2.0	2.0	26	5.6	14.7	33.5	2.5	24.5
	7.0	2.3	2.0	36	5.8	25.8	31.7	2.7	27.3
	24.0	2.6	1.8	55	5.9	38.9	27.6	2.9	30.9
	48.0	2.9	1.7	73	5.9	47.9	25.1	3.2	34.3
	120	3.2	1.6	87	6.0	58.9	22.5	4.0	35.5
	240	3.5	1.6	93	6.2	64.7	20.5	4.3	37.3
	480	3.8	1.5	97	6.1	68.5	19.6	5.8	38.7
CS/Ti alloy	0.0	2.0	2.0	19	5.0	7.3	33.5	1.9	17.3
	0.5	2.6	1.9	22	5.1	7.4	29.4	2.5	18.2
	3.0	3.3	1.8	35	5.2	15.3	28.5	3.1	19.9
	7.0	3.9	1.7	47	5.4	33.7	26.3	3.7	26.9
	24.0	4.5	1.7	67	5.5	61.8	25.1	4.2	33.7
	48.0	5.1	1.6	85	5.6	64.7	21.8	4.9	34.1
	120	5.7	1.5	93	5.7	78.7	20.0	5.5	34.5
	240	6.4	1.5	101	5.7	86.3	19.7	6.7	35.6
	480	7.0	1.4	109	5.8	95.2	18.5	7.6	35.9
CS/TiO2/Ti alloy	0.0	2.1	1.7	27	4.5	7.9	30.1	2.3	23.1
	0.5	2.7	1.5	38	4.7	21.9	27.0	—	18.6
	3.0	3.5	1.3	49	4.8	29.6	25.5	—	20.9
	7.0	4.1	1.2	60	4.9	42.7	23.3	—	24.3
	24.0	4.6	1.1	80	4.9	74.8	20.1	—	28.6
	48.0	5.3	1.1	97	5.0	80.9	19.5	—	37.1
	120	5.9	1.0	100	5.1	99.4	18.2	—	38.3
	240	6.5	0.9	112	5.2	190	17.9	—	39.1
	480	7.2	0.8	120	5.3	224	16.8	—	39.3
CS/TiO2/HA/Ti alloy	0.0	2.5	1.6	67	4.2	8.7	28.5	2.7	22.9
	0.5	3.0	1.4	78	4.4	34.0	26.3	—	36.3
	3.0	3.8	1.2	82	4.6	41.7	23.6	—	26.9
	7.0	4.3	1.0	90	4.7	55.8	21.2	—	29.5
	24.0	4.8	0.8	96	4.8	84.6	18.1	—	33.9
	48.0	5.6	0.7	102	4.8	149	16.5	—	39.8
	120	6.1	0.7	111	4.9	215	16.0	—	45.7
	240	6.7	0.6	123	5.0	314	15.7	—	49.1
	480	7.3	0.5	126	5.0	504	15.3	—	34.7

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Bode plots (Fig. 6a and b) for the CS coated Ti alloy, after immersion for 20 days in PBS at 37 °C, show that the phase diagram was very broad due to the formation of a smooth compact surface of CS, where CS has high adsorption properties.¹⁷ The phase angle maximum reaches nearly 82°. The impedance value increases with increasing time, with higher values (7.0 MΩ cm²) than those of the bare Ti alloy (3.8 MΩ cm²) after 20 days of immersion in PBS. The same model in Fig. 5 was used to fit the data. The Warburg impedance proves that the corrosion reaction is controlled by diffusion phenomena. Fitting data is given in Table 1.

Fig. 6 Bode plots of CS coated Ti–6Al–4V alloy in PBS with immersion time (a) 0–24 h; (b) 2–20 days, at 37 °C.

After incorporation of TiO₂ nanoparticles into the CS matrix, higher impedance values were obtained indicating higher corrosion resistance than both the bare or chitosan coated Ti alloy due to TiO₂ nanoparticles increasing the film strength. Bode plots (Fig. 7a and b) of the CS/TiO₂ coat reveal higher phase angles approaching 85° and higher capacitive behavior; reflecting the improvement of the compact passive film.¹⁸ TiO₂ nanoparticles may fill the pores of the porous outer layer leading to an improvement in the surface film.³ The model used is shown in Fig. 8 which is the same as in Fig. 5 without Warburg impedance indicating that only charge transfer processes control the mechanism of the reaction. Thus, adding TiO₂ nanoparticles changes the mechanism by changing the shape of the Bode plots. TiO₂ nanoparticles increase the thickness of the film formed on the Ti alloy surface by filling the pores in the outer porous layer. This film has higher resistance values, 7.2 MΩ cm², than the chitosan coated film, 7.0 MΩ cm², as shown in Table 1. Both coats have higher resistance values than the bare alloy (3.8 MΩ cm²).

Fig. 7 Bode plots of CS/TiO₂ coated Ti–6Al–4V alloy in PBS with immersion time (a) 0–24 h; (b) 2–20 days, at 37 °C.

Fig. 8 Three time constant equivalent circuit.

Finally, Fig. 9a and b show Bode plots of the nano-composite coat CS/TiO₂/HA after immersion for 20 day in PBS at 37 °C. They show the same trend and mechanism as the CS/TiO₂ nano-coat with higher impedance values and a maximum phase angle near 85° like CS/TiO₂. The broadness of the Bode plot points to a highly stable and formed passive film. According to the proposed model (Fig. 8), the passive film consists of three layers.

Fig. 9 Bode plots of CS/TiO₂/HA nano-coated Ti–6Al–4V alloy in PBS with immersion time (a) 0–24 h; (b) 2–20 days, at 37 °C.

Results of the fitting data are given in Table 1 for all coats. They show that the protection efficiency is mainly due to the inner layer for all coats. A novel CS/TiO₂/HA nano-coat gives a higher inner barrier resistance (R₁) value of 7.3 MΩ cm² compared to 3.8 MΩ cm² for the bare alloy.

From all the previous obtained results, a simple mechanism for how the coat works has been proposed. Chitosan is a biodegradable linear polysaccharide consisting of β-(1–4)-linked D-glucosamine and N-acetyl-D-glucosamine units. It has high adsorption properties¹⁷ on the Ti alloy surface through the lone pair of electrons from the oxygen and nitrogen atoms forming chitosan. This property increases alloy resistance when coated with chitosan over the bare alloy. Titanium oxide has physical/chemical features which are closer to ceramics than to metals.¹⁹ On adding TiO₂ nanoparticles into the chitosan matrix, the bonding strength of the film and its corrosion resistance improve due to their high biocompatibility, and high corrosion and erosion resistance.¹⁵ HA has a low intrinsic strength with high dissolution rate and low stability in biological solutions that hinder its application as a long bone substitute. Its addition with TiO₂ improves its stability and bonding strength with the Ti alloy preventing its corrosion. The bonding strength improvement is the result of the high chemical affinity of TiO₂ toward HA and the Ti alloy.¹⁶ Also, the TiO₂/HA composite in the CS matrix displays excellent adsorption performance.^15,17 So, TiO₂ and HA insertion into the CS matrix improves the bonding strength and coat stability with high adsorption properties to the alloy surface giving the highest protection for the tested alloy by providing a highly compact surface layer.

Generally, TiO₂ may be adsorbed on HA in the chitosan matrix through positively charged calcium ions in HA forming calcium titanate CaTiO₃ as follows:²⁰

Ca₁₀(PO₄)₆(OH)₂ + TiO₂ → 3Ca₃(PO₄)₆ + CaTiO₃ + H₂O (1)

Adsorption of calcium ions on the titanium alloy surface in biological fluids (PBS) might increase its bone biocompatibility.¹⁹ A TiO₂ coating improves calcium phosphate precipitation on the alloy surface²¹ as observed from eqn (1), which is important for biomedical application and implantation. Calcium phosphate exists in the body as amorphous calcium phosphate and as crystalline HA which is a major component of bone.²²

So, this novel synthesized nano-coat including a HA layer is expected to enhance the bioactivity and osteoconductivity of the Ti alloy as an implant.¹⁶

Fig. 10 shows the variation with time of the total resistance (R_T) for the four electrodes in PBS, at 37 °C. It shows the same trend as the open circuit potential measurements with the same order as follows: CS/TiO₂/HA > CS/TiO₂ > CS > bare Ti alloy.

Fig. 10 Variation with time of total resistance (R_T) for the four electrodes in PBS at 37 °C.

The protection efficiency for the three studied coats was calculated using the following equation:^10,23

where R⁰_T and R_T are the total resistances for both the uncoated and nano-coated Ti alloy, respectively. The results are given in Table 2. It was found that CS/TiO₂/HA gives the highest PE% reaching 92.11% and 89.47% for CS/TiO₂ and the lowest value of 84.21% for the CS coat.

Table 2 PE% of coated Ti alloy in PBS with immersion time, at 37 °C

Electrodes	Time/h	PE%
CS/Ti alloy	0.0	53.85
	0.5	62.50
	3.0	65.00
	7.0	69.57
	24.0	73.08
	48.0	75.86
	120	78.13
	240	82.86
	480	84.21
CS/TiO2/Ti alloy	0.0	61.54
	0.5	68.75
	3.0	75.00
	7.0	78.26
	24.0	76.92
	48.0	82.76
	120	84.38
	240	85.71
	480	89.47
CS/TiO2/HA/Ti alloy	0.0	92.31
	0.5	87.50
	3.0	90.00
	7.0	86.96
	24.0	84.62
	48.0	93.10
	120	90.63
	240	91.43
	480	92.11

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Generally, the novel CS/TiO₂/HA nano-coat has the highest corrosion resistance with the highest PE% confirming the OCP measurements.

3.3. Potentiodynamic polarization measurements

Potentiodynamic polarization analysis was performed to get further information about the corrosion properties of the formed coats, where the corrosion rate is proportional to the corrosion current density. Potentiodynamic polarization measurements were done in the range from −1.5 V to 2.0 V vs. SCE (Fig. 11), at a scan rate of 1.0 mV s⁻¹ in phosphate buffer solution after reaching a steady state potential value, at 37 °C. The polarization parameters (i_corr, E_corr, β_a and β_c) are given in Table 3. The corrosion potential (E_corr) and corrosion current density (i_corr), equivalent to corrosion rate, are given by the intersection of the Tafel lines.^24,25 Anodic and cathodic Tafel constants are symbolized, respectively, as β_a and β_c. They are calculated from the anodic and cathodic slopes of the Tafel plot at the points after E_corr by ±50 mV using a computer least-squares method due to the degree of nonlinearity in the Tafel slope plot.²⁴

Fig. 11 Polarization scans for nano-coated and uncoated Ti alloy after immersion for 20 days in PBS at 37 °C.

Table 3 Corrosion parameters of bare and coated Ti alloy in PBS after immersion for 480 hour, at 37 °C

Electrodes	E corr/mV	i corr/µA cm^−2	β a/mV dec^−1	−βc/mV dec^−1	PE%
Bare Ti alloy	−348	15.8	11.1	9.8	—
CS/Ti alloy	−304	2.50	10.5	8.4	84.2
CS/TiO2/Ti alloy	−188	0.79	9.7	7.9	95.0
CS/TiO2/HA/Ti alloy	−120	0.41	8.5	6.1	97.4

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The E_corr value for the four tested alloys revealed a shift towards more positive values as obtained from the open circuit potential in the following order: CS/TiO₂/HA (−120 mV) > CS/TiO₂ (−188 mV) > CS (−304 mV) > bare Ti alloy (−348 mV). Also, the i_corr values increased in the same order, indicating that the CS/TiO₂/HA nano-coat has the lowest i_corr value (0.41 µA cm⁻²) and the bare electrode has the highest i_corr value (15.8 µA cm⁻²) with the highest corrosion rate. However, adding chitosan decreased the corrosion rate due to it being a macromolecule adsorbed well on the surface through electron pairs.²⁶ Adding TiO₂ into the chitosan matrix decreases the corrosion rate by increasing the film strength and then adding HA gives the lowest corrosion rate. This is due to CS increasing the adsorption of both the TiO₂ and HA nanoparticles to the Ti alloy surface. The TiO₂ nanoparticles increase the adhesion strength between the HA nanoparticles in the chitosan matrix and the Ti alloy surface.²⁷ This leads to an increase in film stability, strength and adsorption on the alloy surface. Also phosphate buffer forming ions helps in decreasing the corrosion rate of the Ti alloy.²⁸

Finally, the CS/HA/TiO₂ nanostructured dense coating acts as an effective barrier to the transport of electrons and ions between the Ti alloy and the PBS solution.²² So, this novel synthesized nano-coat significantly improved the corrosion resistance of the Ti alloy surface.

The protection efficiency (PE%) is also given in Table 3 and was calculated from the following equation:^10,18

where i_uncoated and i_coated are the uncoated and nano-coated corrosion current densities, respectively. It was found that the PE% for CS/TiO₂/HA was the highest reaching 97.4% whereas it only reached 95% for CS/TiO₂ and 84.2% for CS. These results are in the same trend with those obtained from the impedance measurements. Also the PE% obtained by impedance measurements is near to that obtained from polarization measurements.

Thus, the polarization results confirm the impedance, SEM, EDX and open circuit potential measurements.

3.4. Antibacterial activity measurements

Fig. 12a and b report the inhibition zone test results for both the bare Ti alloy and the novel nano-coated one. The inhibition zone (Fig. 12a) for the bare sample to Staphylococcus aureus shows no bacteriostasis, while for the CS/TiO₂/HA nano-coated sample (Fig. 12b), the inhibition zone is recognizable and obvious antimicrobial ability is shown. This confirms that this novel nano-composite coat has antibacterial efficiency by its ability to adsorb bacteria.¹⁵ This means that it has excellent resistance against Staphylococcus aureus on the bone and can be used as an effective biomedical implant. Effective physiological bone integration in the absence of bacterial contamination is crucial for an effective orthopaedic implant. It is important to obtain antibacterial surfaces and this has been shown with the novel synthesized CS/TiO₂/HA nano-coat that can be considered to be good in antibacterial applications.¹⁵

Fig. 12 Inhibition zones of (a) bare sample and (b) CS/TiO₂/HA nano-coated sample.

This is confirmed by the high corrosion resistance of the synthesized CS/TiO₂/HA nano-coat as calculated from all the used electrochemical and surface techniques.

4. Conclusions

This work was done on TiO₂ and HA nanoparticles inserted into a chitosan (CS) matrix. This novel synthesized nano-coat on a Ti alloy gives rise to an organic/inorganic non-toxic coat. The following conclusions were drawn:

(1) SEM and EDX analysis of the coated films indicated the formation of a stable compact and smooth passive film of TiO₂ and HA incorporated into a CS matrix.

(2) Open circuit potential measurements showed that the CS/TiO₂/HA nano-coat demonstrates a higher stability than the other coats with the lowest negative OCP. The order of the OCP was as follows: CS/TiO₂/HA (−120 mV) > CS/TiO₂ (−188 mV) > CS (−304 mV) > bare Ti alloy (−348 mV). This also indicates that the bare alloy has the highest negative potential leading to highest corrosion rate.

(3) An electric equivalent circuit with three time constants was modeled for the four tested electrodes.

(4) The CS/TiO₂/HA nano-coat has the highest corrosion resistance of 7.3 MΩ cm² due to the complete coverage of the Ti alloy surface protecting it from corrosion.

(5) The lowest corrosion current density of 0.41 µA cm⁻² was observed for the CS/TiO₂/HA nano-coat with the protection efficiency reaching 97.4% compared to other coats due to the enhancement of the protective properties of the passive layer for this coat.

(6) The CS/TiO₂/HA nano-coat has a high antibacterial activity compared to the bare alloy confirming its high protection for bone.

Generally and finally the SEM, EDX and open circuit potential measurements are in good agreement with the polarization and impedance measurements. Also, this novel coat is synthesized from both organic and inorganic materials. It is an important coat to be applied for implants in the human body.

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