Mineralized

Article pubs.acs.org/IECR

Carbon Nanotubes/Carboxymethyl Chitosan/Mineralized Hydroxyapatite Composite Coating on Ti-6Al-4V Alloy for Improved Mechanical and Biological Properties D. Gopi,*,†,‡ S. Nithiya,† E. Shinyjoy,† D. Rajeswari,†,§ and L. Kavitha∥ †

Department of Chemistry, ‡Centre for Nanoscience and Nanotechnology, and §Department of Physics, Periyar University, Salem 636 011, Tamilnadu, India ∥ Department of Physics, School of Basic and Applied Sciences, Central University of Tamilnadu, Thiruvarur 610 101, Tamilnadu, India ABSTRACT: Hydroxyapatite (HAP) is the most suitable nontoxic, biocompatible material increasingly used for bone implant coatings. However, its brittle nature is a major obstacle for such applications and this leads to the focus on developing composite coatings with the incorporation of various biopolymers and reinforcing material. In this study, mineral-substituted hydroxyapatite (M-HAP) and carboxymethyl chitosan (CMC), a biopolymer, are made into a composite (CMC/M-HAP) for enhanced biological properties of HAP. Furthermore, carbon nanotubes (CNTs) are incorporated in the composite to improve the mechanical and anticorrosive properties of HAP. The present work investigates the development of CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy for improved biological and mechanical properties, which is anticipated to be the most suited alternative material for orthopedic implants. polymer and it has many applications in medicine.19 Since there is no single material available to fulfill all the necessary requirements for biomedical applications, development of composites as an alternative for bone repair has become mandatory.20 The mechanical properties of chitosan/HAP composite can be further improved by the addition of suitable reinforcing material. Carbon nanotubes (CNTs), because of their small dimensions and high aspect ratio, exhibit excellent physical and chemical properties.21,22 No other material can compared with the excellent mechanical properties of CNTs, which make them an outstanding reinforcing material for composite formation23,24 without diminishing bioactivity.25 Poor dispersibility of CNTs has been the greatest obstacle to their use in biomedicine. Many functionalization routes have been developed in recent years to solubilize CNTs and to improve their dispersibility. Furthermore, the formation of composites with CNTs as an excellent reinforcement material finds applications in major load-bearing devices. Moreover, the bioactivity of HAP is not affected by the incorporation of CNTs, thus opening up a wide range of clinical applications of this material.26 A composite of CNTs/CMC/M-HAP is anticipated to provide a favorable combination of mechanical and biological properties, which is an essential requirement in the field of biomedical science. Coating of this composite on implant materials, like surgical-grade stainless steel and titanium and its alloys, improves the biological properties and also the corrosion resistance of the implant.27−29 Several methods such as sol−gel, pulsed laser deposition, electrophoretic method, etc.,30−34 have been reported for

1. INTRODUCTION Hydroxyapatite [Ca10(PO4)6OH)2, HAP] is a nontoxic bioactive ceramic increasingly used as a biocompatible coating material that encourages the adaptation of an implant into the human body by forming a direct bond with living tissue. Being a prime constituent of human bone, HAP shows excellent bioactivity, osteoconductivity, and biocompatibility.1 HAP is able to promote bone growth and adhesion on the surface of the implant during the early stages of implantation.2,3 Several studies have reported that the introduction of small quantities of minerals such as strontium (Sr 2+), barium (Ba 2+), magnesium (Mg2+), zinc (Zn2+), etc., into the HAP structure improved the structural stability and biocompatible properties of HAP.4−6 Among these ions, the incorporation of minerals such as Sr2+, Mg2+, and Zn2+ has been of great interest for the biological process due to their significant properties.7 Strontium has a beneficial effect on bone, and strontium-containing HAP was designed as a filling material to improve the biocompatibility of bone cement. In vitro studies demonstrated that Sr enhanced the replication of osteoblast cells and inhibited osteoclast activity.8,9 Mg2+ substitution in HAP has been the subject of extensive research because of its ability to develop artificial bone and other medical applications.10,11 Mg2+ plays an important role in preventing osteoporosis in human bone.12 Zn2+ is naturally present in bone and also stimulates bone growth as well as bone mineralization.13 It has a direct effect on osteoblastic cells in vitro and an inhibitory effect on osteoclastic bone resorption in vivo,14 in addition to its antimicrobial activity.15 Polymers combined with HAP are useful for potential applications in bone repair and regeneration.16 Carboxymethyl chitosan (CMC), a chitosan-derived polymer, has attracted much interest in a wide range of biomedical applications.17,18 Though chitosan is nonsoluble, CMC is a water-soluble © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7660

November 18, 2013 March 30, 2014 April 13, 2014 April 14, 2014 dx.doi.org/10.1021/ie403903q | Ind. Eng. Chem. Res. 2014, 53, 7660−7669

Industrial & Engineering Chemistry Research

Article

Figure 1. Mechanism for formation of CNTs/CMC/M-HAP composite.

6H2O), zinc chloride (ZnCl2), dipotassium hydrogen phosphate (K2HPO4), chitosan, sodium hydroxide, isopropyl alcohol, acetic acid, and single-walled CNTs with a diameter of 1.2−1.5 nm, purchased from Aldrich Chemical Co. (Aldrich, India), were used for the development of CNTs/CMC/MHAP composite coating on Ti-6Al-4V alloy by electrophoretic deposition. All chemicals were of analytical grade and were used as received, and deionized water was used throughout the experiment. 2.2. Synthesis of CMC and M-HAP and Functionalization of CNTs for Formation of Composite. 2.2.1. Synthesis of CMC. CMC was synthesized according to the method followed earlier37 with slight modification. In this preparation, 4 g of chitosan was added to 100 mL of isopropyl alcohol and the mixture was stirred with a magnetic stirrer at room temperature

deposition of HAP coatings onto implant surfaces. Among these, the electrophoretic deposition (EPD) method seems to be more appropriate to produce homogeneous and dense ceramic, polymer, and composite coatings at reduced cost for biomedical applications.35,36 In the present work, we report the synthesis and development of a CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy by EPD to provide enhanced corrosion resistance and mechanical and biological properties, so as to serve as a better candidate for load-bearing applications.

2. MATERIALS AND METHODS 2.1. Materials. Commercially available calcium chloride tetrahydrate (CaCl2·4H2O), strontium chloride hexahydrate (SrCl2·6H2O), magnesium chloride hexahydrate (MgCl2· 7661

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were visually inspected. All experiments were conducted at ambient temperature, and samples were carefully and slowly pulled out of the suspension after EPD. Finally, the samples were dried in air for 24 h and stored in a desiccator at room temperature. 2.7. Surface Characterization. Fourier transform infrared (FTIR) spectra of the composite were recorded on a Nicolet 380 FTIR spectrometer (PerkinElmer) over the frequency range from 4000 to 400 cm−1 with 32 scans and spectral resolution of 4 cm−1. The surface morphology and elemental composition of the composite coatings were examined by scanning electron microscopy (SEM)/energy-dispersive X-ray (EDX) analysis (JEOL JSM-6400). The microstructure of the composite coating was characterized by high-resolution transmission electron microscopy (HRTEM) (JEOL JEM 2100 Co., Tokyo, Japan). Samples for HRTEM analysis were prepared by scraping the CNTs/CMC/ M-HAP composite coating from the Ti-6Al-4V alloy and dispersing it in ethanol, followed by sonication for 10 min. Then a drop of suspension was deposited on a copper-coated carbon grid with 200 mesh, and the solvent was allowed to evaporate. X-ray photoelectron spectroscopy (XPS; Omicron NanoTechnology instrument) with a focused monochromatic Al Kα source (1486.6 eV) for excitation was utilized for chemical composition analysis. The electron takeoff angle was 54.7° and the analyzer was operated in constant-energy mode for all measurements. XPS survey spectrum over a binding energy range of 0−1100 eV was acquired with analyzer pass energy of 50 and 20 eV for high-resolution elemental scans. The vacuum pressure was around 3.5 × 10−10 mbar during spectral acquisition. Data analysis was carried out with EIS-Sphera software provided by the manufacturer. The hydrocarbon peak maximum in the C 1s spectra was set as 284.6 eV to reference the binding energy scales for the samples. Intensity ratios were converted into atomic concentration ratios by using the sensitivity factors proposed by the manufacturer. Microstructural investigation and elemental analysis of the composite coatings were examined by high-resolution scanning electron microscopy (HRSEM, JEOL JSM-6400, Japan) and energy-dispersive X-ray analysis (EDX, Horiba 7021-H, England), respectively. Prior to SEM imaging at 30 kV, the samples were sputter-coated with gold in order to obtain sufficient conductivity on the surface and to avoid charging of the surface during SEM. 2.8. Electrochemical Evaluation of Composite Coatings. Electrochemical measurements were carried out through potentiodynamic polarization to assess the anticorrosive characteristics of the coatings. A saturated calomel electrode (SCE), platinum electrode, and Ti-6Al-4V alloy were used as the reference, counter, and working electrode, respectively. Simulated body fluid (SBF) solution with ion concentration nearly equal to that of human blood plasma was used as electrolyte medium. This solution was prepared according to the protocol suggested by Kokubo and Takafama.40 Potentiodynamic polarization studies were measured at a scan rate of 1 mV·s−1 in the potential range between −200 and −2000 mV. The obtained data was recorded by use of internally available software, and each experiment was repeated three times to check reproducibility. 2.9. Mechanical Properties of Composite Coatings. Adhesion strength between the composite coating and substrate was evaluated by the standard scratch tester and

for 5 h. The suspension was then transferred to a 500 mL round-bottomed flask; 100 mL of aqueous NaOH (50%) was added and the mixture was refluxed at 85 °C for 4 h. Then 100 mL of aqueous monochloroacetic acid was added into the above mixture within 20 min. The mixture was heated under stirring at 65 °C for a further 12 h, and the reaction mixture was then neutralized by use of hydrochloric acid solution (4 M). After removal of the undissolved residue by filtration, the resultant CMC was precipitated by adding methyl alcohol. The product was filtered and washed several times with a CH3OH/ H2O mixture (1:1) and dried under vacuum. 2.2.2. Synthesis of M-HAP. M-HAP synthesis was carried out with 0.05 M CaCl2·4H2O, SrCl2·6H2O, MgCl2·6H2O, and ZnCl2 and 0.03 M K2HPO4 aqueous solution. The synthesis procedure was adopted from our earlier report.7 2.2.3. Functionalization of CNTs. Functionalization of CNTs was performed to improve the dispersion stability and better interactions with composite. The functionalization of CNTs was carried out in accordance with our previous work.38 2.3. Synthesis of Composite. 2.3.1. Synthesis of CMC/MHAP Composite. Composite of CMC/M-HAP was prepared through the ultrasonication process by use of an ultrasonicator [EN-60US (Microplus), frequency 28 kHz, 150 W) with a ratio of 1.5 g of M-HAP and 0.5 g of CMC in 40 mL of ethanol− water solvent. This mixture was ultrasonicated for 2 h to ensure good dispersion. It was then filtered, washed, dried at 110 °C for 12 h, and ground into a fine powder. 2.3.2. Synthesis of CNTs/CMC/M-HAP Composite. The CNTs/CMC/M-HAP composite was prepared from known quantities of CMC, O-CNTs, and M-HAP in 40 mL of ethanol−water mixture. This mixture was stirred and ultrasonicated for 2 h to ensure good dispersion. It was then filtered, washed, dried at 80 °C for 12 h, and ground into a fine powder. The mechanism for formation of CNTs/CMC/M-HAP composite for EPD is clearly illustrated in Figure 1. 2.4. Specimen Preparation. Ti-6Al-4V alloy, with composition (wt %) 5.7 Al, 3.85 V, 0.18 Fe, 0.038 C, 0.106 O, 0.035 N, and balance titanium, was used in the present study. Pieces of Ti-6Al-4V alloy with a size of 10 × 10 × 3 mm were cut and embedded in epoxy resin, leaving an area of 1 cm2 for exposure to the solution, which was used as the substrate for the EPD of CNTs/CMC/M-HAP composite. Prior to EPD, the samples were abraded with different grades of SiC emery papers from 400 to 1200 grit, washed with distilled water, degreased with acetone, and then dried at room temperature. 2.5. Piranha Treatment. The Ti-6Al-4V alloys after polishing were sonicated for 45 min in 70% isopropyl alcohol. Followed by sonication, the specimens were etched for 15 min in a mixture of 7:3 molar ratio concentrated H2SO4 and 35% H2O2 (piranha solution).39 After etching, the specimens were washed with deionized water and dried in air. The piranhatreated Ti-6Al-4V alloy is further used for coating with composite. 2.6. Electrophoretic Deposition of CNTs/CMC/M-HAP Composite on Ti-6Al-4V Alloy. Electrophoretic deposition experiments were carried out at room temperature under dc voltage. Ti-6Al-4V alloy was used as the cathode and platinum electrode was used as the anode. Electrophoretic deposition was carried out by setting a constant voltage of 30 V and the deposition was performed for 7 min. The distance between the two electrodes was 3 cm. These conditions were obtained by an optimization process based on a trial-and-error approach to attain deposits of satisfying thickness and uniformity, which 7662



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indentation on a Universal Instron mechanical testing system (Instron 5565, Instron Co.) according to ASTM F 1044-05 standard.41 The specimens were subjected to tests at a constant cross-head speed. 2.10. In Vitro Biocompatibility Studies. 2.10.1. Cell Culture. HOS MG63 cells were obtained from the National Centre for Cell Science (NCCS), Pune, India, and were cultured in minimal essential medium (Hi Media Laboratories) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 units·mL−1), and penicillin (100 units·mL−1) (Cistron Laboratories). The cell culture was then incubated under a humidified atmosphere (CO2) at 37 °C. The samples under examination were sterilized in an autoclave at 120 °C for 2 h and placed in 24-well cell culture plates. 2.10.2. Cell Viability Test. Cell viability was determined by MTT [3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrozolium bromide] assay.42 Cells at a density of 1 × 105/well were seeded on the samples in 24-well culture plates. The culture medium was replaced with new medium every day. After 48 h of incubation, the samples were removed from the respective wells and the wells were washed with phosphate-buffered saline (PBS, pH = 7.4). Only those cells that are adherent to the well walls were found viable and incubated with 0.5% MTT solution. The viable cells reduce the MTT into insoluble formazan precipitate by mitochondrial succinic dehydrogenase. After 4 h of incubation, 0.1% dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. After these procedures, the absorbance of the content of each well was determined at 570 nm with a UV spectrophotometer. Cell viability (percent) related to the control wells containing cell culture medium without the samples was calculated from the average of five replicates as

Figure 2. FTIR spectra of (a) O-CNTs, (b) chitosan, (c) CMC, and (d) CNTs/CMC/M-HAP composite.

Table 1. Characteristic Infrared Peaks of O-CNTs, Chitosan, CMC, and CNTs/CMC/M-HAP Composite

%cell viability = [A]test /[A]control × 100

2.10.3. Cell Adhesion Test. HOS MG63 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin−streptomycin cocktail. The samples used for cell adhesion were sterilized in an autoclave (121 °C, 0.1 MPa pressure for 15 min), and subsequently, the cells were seeded on the samples at a density of 5 × 105 mL−1. The seeded test sample was incubated in a CO2 incubator under standard culture conditions. The culture medium was aspirated after a 2 day interval, and fresh culture medium was added into each well. After the stipulated time period of 36−48 h, the samples were washed twice with phosphate-buffered saline (1× PBS, pH 7.4). For cell morphology observation, the HOS MG63 cells attached to the samples were fixed with 2% glutaraldehyde for 1 h at room temperature, followed by dehydration with a series of ethanol/water solution for 10 min twice. Then 0.5 mL of hexamethyldisilazane (HMDS, Hi Media) was added to each well to preserve the original morphology of the cells. The samples were coated with gold (for conduction) before observation by SEM.

peak (cm−1)

3. RESULTS AND DISCUSSION 3.1. Surface Characterization. Fourier transform infrared spectroscopy is an indispensable technique for identification of functional groups present in the composite. Figure 2 shows the FTIR spectra for O-CNTs, chitosan, CMC, and CNTs/CMC/ M-HAP, and their corresponding predominant characteristic IR peaks are given in Table 1. Figure 2a shows the characteristic peaks of O-CNTs as reported by Gopi et al.38 Figure 2b shows the characteristic peaks of chitosan, which are in good

961 473 1030, 1092 574, 603 3569, 632 3437, 1638 1738

assignment O-CNTs38

3500 1750 1094 3440 2872 1327 1140, 1080 1025 3455 2922, 2874 1600 1327 1087 1409, 1570 1150

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OH stretching CO stretching C−O stretching Chitosan43 O−H and N−H stretching C−H stretching C−N stretching primary amine stretching pyranose (C−O−C) stretching CMC44 OH stretching C−H stretching N−H bending C−N stretching C−O stretching symmetric and asymmetric COO− bridge O stretching CNTs/CMC/M-HAP Composite symmetric stretching of phosphate (υ1) O−P−O bending mode (υ2)45 asymmetric stretching of phosphate (υ3) bending mode of phosphate (υ4) stretching and bending mode of surface OH− group of HAP46 stretching and bending mode of adsorbed H2O molecule47 amide linkage of −CO−NH−48



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Figure 3. SEM micrographs of (a) untreated Ti-6Al-4V alloy, (b) piranha-treated Ti-6Al-4V alloy, (c) CNTs/CMC/M-HAP composite coating on untreated Ti-6Al-4V alloy, (d) CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy, and (e) CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy. (f) Elemental composition of CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy.

agreement with the reported literature.43 Figure 2c confirms the formation of CMC with absorption peaks at 1409 (sym COO−), 1570 (asymm COO−), and 1150 cm−1 (bridge O stretching).44 The spectrum for the composite (Figure 2d) shows the characteristic peaks of O-CNTs, CMC, and MHAP.45−47 Strong absorption peaks appearing at 1738 cm−1 (amide group) indicate that −COOH group of the CNTs reacts with the NH2 group of CMC and converts it into the amide (−CO−NH) group. This peak is well evident for the formation of the composite.48 The surface morphologies of untreated alloy, piranha-treated alloy, CNTs/CMC/M-HAP composite coating on untreated alloy, CMC/M-HAP composite coating on piranha-treated alloy, and CNTs/CMC/M-HAP composite coating on piranhatreated Ti-6Al-4V alloy are shown in Figure 3a−e. Before surface treatment, the surface of Ti-6Al-4V alloy revealed no distinct features (i.e., surface was undisturbed) (Figure 3a). For the sample treated with piranha solution, the morphology (Figure 3b) exhibits the presence of a large number of minute pores on the surface with aggregation in a few places, which paves the way for strong and adherent coating on the surface of

Figure 4. HRTEM image of CNTs/CMC/M-HAP composite coating.

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Figure 5. (a) XPS survey spectrum of CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy, and deconvolution spectra of (b) Ca, (c) Sr and P, (d) O, (e) C, and (f) N.

the Ti-6Al-4V alloy. According to the literature,49 homogeneous distribution of the components is the most critical step in performing EPD. If the electrolyte for deposition contains coagulated particles, then the deposited layer will be inhomogeneous, which results in surface cracks within the coating layer. Thus, in order to create a homogeneous suspension of CNTs and CMC/M-HAP, we have preassembled them to form a single composite that was later deposited onto the substrate. Figure 3c shows the CNTs/CMC/M-HAP

composite coating on untreated Ti-6Al-4V alloy, which showed the existence of aggregates of granules of the composite material. The coating appears to be loosely arranged over the untreated Ti-6Al-4V alloy. When the CMC/M-HAP composite is deposited on piranha-treated Ti-6Al-4V alloy (Figure 3d), the coating appears to be smooth with few micropores over the surface. For the CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V, the SEM image (Figure 3e) revealed compact packing of the composite with micropores and dense 7665



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Table 2. Binding Energy and Atomic Percentage of Characteristic Peaks of XPS Spectra of CNTs/CMC/M-HAP Compositea

a

core level

binding energy (eV)

at. %

Ca 2p1/2 Ca 2p3/2 P 2s P 2p O 1s C 1s N 1s Sr 3d5/2 Mg 2p Mg 2s Zn 2p3/2 Zn 2p1/2

350.7 347.5 133.2 190.2 530.8 284.6 400.4 133 ± 0.5 49.9 88.7 1021.6 1044.5

2.086 0.633 37.274 45.992 1.137 0.231 11.337

Figure 7. Bar diagram showing percent cell viability of HOS MG63 cells on composite coating.

3.726

Spectra are shown in Figure 5.50−54.

Figure 6. Potentiodynamic polarization curves for pristine Ti-6Al-4V alloy, CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy, CNTs/CMC/M-HAP composite coating on untreated Ti-6Al4V alloy, and CNTs/CMC/M-HAP composite coating on piranhatreated Ti-6Al-4V alloy.

Figure 8. Optical images of (a) control, (b) CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy, and (c) CNTs/CMC/MHAP composite coating on piranha-treated Ti-6Al-4V alloy at 7 days of incubation.

Table 3. Electrochemical Parameters of Untreated and Coated Ti-6Al-4V Alloys in Simulated Body Fluid sample untreated Ti-6Al-4V alloy CMC/M-HAP coating on piranha-treated Ti-6Al4V alloy CNTs/CMC/M-HAP coating on pristine Ti-6Al4V alloy CNTs/CMC/M-HAP coating on piranha-treated Ti-6Al-4V alloy

Ecorr (mV)

icorr (μA·cm−2)

−12 82

1.29 0.87

143

0.63

230

0.48

In order to confirm the presence of CNTs in the CNTs/ CMC/M-HAP composite, high-resolution TEM analysis was performed, and the micrograph is shown in Figure 4. The tubular structure observed in the figure represents the presence of CNTs in the composite of CNTs/CMC/M-HAP. Moreover, the spherical particles of CMC/M-HAP are found to be closely attached to the surface of CNTs. Hence, the presence of CNTs in the CNTs/CMC/M-HAP composite coating could be confirmed. A typical survey XPS spectrum for CNTs/CMC/M-HAP composite-coated Ti-6Al-4V alloy is shown in Figure 5a, and the corresponding deconvolution spectra are shown in Figure 5b−f. The survey spectrum identified Ca, P, Sr, Mg, Zn, C, O, and N as the major constituents of the composite coating on Ti-6Al-4V alloy substrate.50−54 Binding energies of the characteristic peaks and quantified atomic percent elemental composition of the as-formed composite on Ti-6Al-4V alloy are reported in Table 2. All the XPS data support the formation of CNTs/CMC/M-HAP composite.

foamlike structure covering the entire surface. The foamlike structure could afford an appropriate environment for cells, which benefits adhesion and proliferation on the surface of the composite coating. The EDX spectrum showing the constituent elements of the CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy is presented in Figure 3f. The spectrum shows the presence of Ca, P, O, Sr, Mg, Zn, C, and Ti. This result reveals the existence of CNTs, CMC, and MHAP in the composite. 7666



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adhesion strength of HAP and CMC/M-HAP coating on surface-treated alloy, respectively, and the CNTs/CMC/MHAP composite coating on untreated alloy. This demonstrates the role of CNTs as a reinforcing material in composite coating and also the role of piranha treatment on the Ti-6Al-4V alloy prior to the coating. 3.4. In Vitro Cytotoxicity Studies with HOS MG63 Cells. 3.4.1. MTT Assay Test. The viability of HOS MG63 cells on CMC/M-HAP and CNTs/CMC/M-HAP composite coatings on piranha-treated Ti-6Al-4V alloy was determined by MTT assay. Absorbance at 570 nm is directly proportional to the number of living cells in the culture. Cell viability is calculated for the composite coating on piranha-treated Ti-6Al4V alloy with respect to control. The percent cell viability (125 μg·mL−1) of both composite coatings at 1, 4, and 7 days of culture is shown in Figure 7. Both composite coatings exhibited appreciable cell viability similar to the control group. The CNTs/CMC/M-HAP composite coating on piranha-treated alloy showed increased cell viability (from 89.9% ± 0.7% to 96.7% ± 0.5% for 1 to 7 days of incubation), whereas the CMC/M-HAP coating on piranha-treated Ti-6Al-4V alloy showed an increase from 82.8% ± 0.4% to 94.6% ± 0.5% viability for 1 to 7 days. Therefore, the CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy exhibited higher/greater viability of cells at 7 days of incubation, which may be due to the presence of mineral ions in HAP; also, this effect clearly explains the nontoxicity of CNTs present in the CNTs/CMC/M-HAP composite.55 Figure 8 shows the optical microscopic results for control, CMC/M-HAP, and CNTs/CMC/M-HAP composite coatings in 24-well cultured plates after the incubation period of 7 days. It is clear from the figure that a number of cells were found to be viable in the CMC/M-HAP composite coating, as seen in Figure 8c. Also, the addition of CNTs in the CMC/M-HAP composite (Figure 8c) exhibited cell morphology similar to the control group and the cells were completely spread out, showing the biocompatibility of the CNTs/CMC/M-HAP. This confirms that the addition of reinforcing material like CNTs in the composite does not affect the biocompatibility of the composite. Thus, it is clear from this study that the presence of CNTs as an ideal reinforcement material in the CNTs/CMC/M-HAP composite would impart excellent mechanical properties to the composite at high loading applications without diminishing its bioactivity. Thus, from our findings we have concluded that CNTs do not affect the bioactivity of the composite but promote cell growth. 3.4.2. Cell Attachment. Figure 9 shows a typical SEM image of HOS MG63 cells after 7 days of culture on the surface of the CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy. It was observed from the figure that cells were attached and spread over the entire surface. Typically, the cells were flattened and few pseudopods were seen, which indicates good livelihood of the HOS MG63 cells on the CNTs/CMC/ M-HAP coating on piranha-treated Ti-6Al-4V alloy. The proliferation of cells requires the presence of growth factors in the medium, which is provided by either serum or purified proteins.55 The mineral-substituted (Sr, Mg, and Zn) HAP and the CMC present in the composite coating provided the necessary nutrients for the growth of cells,7,56−59 whereas the CNTs remarkably improved the biological cellular response to the coating.60 Hence, this cell culture result shows that CNTs/ CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy have good biocompatibility without any toxicity.

Figure 9. HRSEM micrograph of HOS MG63 cell growth on CNTs/ CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy.

3.2. Potentiodynamic Polarization Measurements. Potentiodynamic polarization curves of untreated alloy, CMC/M-HAP coating on piranha-treated alloy, CNTs/ CMC/M-HAP composite coating on untreated alloy, and CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy in SBF solution are shown in Figure 6. The corrosion potential (Ecorr) and corrosion current density (icorr) values for uncoated and coated samples were determined and are summarized in Table 3. As evidenced from the table, the coatings on Ti-6Al-4V alloy obtained under different conditions showed more positive Ecorr and much lower icorr compared to the untreated Ti-6Al-4V alloy. In particular, Ecorr of CMC/MHAP coating on piranha-treated Ti-6Al-4V alloy was shifted to positive direction, whereas the addition of CNTs in the composite coating on untreated Ti-6Al-4V alloy enhanced the corrosion resistance, which can be evidenced from the obtained Ecorr and icorr values (Table 3). It could be mainly due to the presence of CNTs as an excellent reinforcing material in the composite coating. To further improve corrosion resistance, the same composite (CNTs/CMC/M-HAP) was coated on piranha-treated Ti-6Al-4V alloy, and that exhibited enhanced bioresistive nature compared to the CNTs/CMC/M-HAP coating on untreated Ti-6Al-4V alloy. Thus, potentiodynamic polarization studies showed that the CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy had more positive corrosion potential, which implies that the corrosion resistance of the Ti-6Al-4V alloy was significantly improved by piranha treatment and also due to the presence of CNTs in the CNTs/CMC/M-HAP composite coating. 3.3. Adhesion Strength. The adhesion strength of composite (CMC/M-HAP and CNTs/CMC/M-HAP) coatings on piranha-treated Ti-6Al-4V alloy was evaluated. Adhesion strength is based on the combination of adhesive and cohesive strength of the composite coating.40 The adhesive strength of HAP and CMC/M-HAP composite coatings obtained on piranha-treated Ti-6Al-4V alloy was measured as 15.3 ± 0.9 and 24.2 ± 0.8 MPa, respectively. However, there is an appreciable improvement in the adhesion strength of CNTs/CMC/M-HAP composite coating (29.2 ± 0.8 MPa) on piranha-treated Ti-6Al-4V alloy, which is higher than the 7667



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4. CONCLUSIONS Herein, we demonstrate the synthesis and development of CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy by EPD for improved mechanical and biological properties. The formation of composite (CNTs/ CMC/M-HAP) is preferred over the single material (CNTs, CMC and M-HAP) because the constituents of the composite material are found to complement each other and improve the required properties. Thus, the composite exhibited wonderful properties compared to that of single material. This is well evidenced from the adhesion strength and cell culture results. The presence of CNTs improves the strength of the composite, whereas the biological properties of the composite are enhanced by the presence of CMC and minerals present in the composite. Thus, the CNTs/CMC/M-HAP composite coating on piranha-treated Ti-6Al-4V alloy will serve as an indispensable implant material with improved mechanical and biological properties.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +91 427 2345766. Fax: +91 427 2345124. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.G. acknowledges major financial support from the Indian Council of Medical Research (ICMR) (IRIS ID 2010-08660, grant 5/20/11(Bio)/10-NCD-I), Department of Science and Technology, New Delhi, India (DST-TSD, grant DST/TSG/ NTS/2011/73, and DST-EMEQ, grant SB/EMEQ-185/2013), and Council of Scientific Industrial Research (CSIR, grant 01(2547)/11/EMR-II, dated 12.12.2011). Also, D.G. and L.K. acknowledge the UGC (F.30-1/2013 (SA-II)/RA-2012-14NEW-SC-TAM-3240 and F.30-1/2013(SA-II)/RA-2012-14NEW-SC-TAM-3228) for Research Awards. D.R. acknowledges major financial support from the DST [SR/WOS-A/PS26/2012 (G)].

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