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hydroxyapatite hybrids on surgical grade 316L SS with enhanced corrosion resistance and bioactivity
Accepted Manuscript Title: Electrochemically grown functionalized -Multi-walled carbon nanotubes/hydroxyapatite hybrids on surgical grade 316L SS with enhanced corrosion resistance and bioactivity Authors: S. Arul Xavier Stango, U. Vijayalakshmi PII: DOI: Reference:
S0927-7765(18)30429-6 https://doi.org/10.1016/j.colsurfb.2018.06.058 COLSUB 9448
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-2-2018 28-5-2018 27-6-2018
Please cite this article as: S. Arul Xavier S, U. V, Electrochemically grown functionalized -Multi-walled carbon nanotubes/hydroxyapatite hybrids on surgical grade 316L SS with enhanced corrosion resistance and bioactivity, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.06.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Electrochemically grown functionalized -Multi-walled carbon nanotubes/hydroxyapatite hybrids on surgical grade 316L SS with enhanced corrosion resistance and bioactivity.
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S. Arul Xavier Stango and U. Vijayalakshmi*
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Department of Chemistry, School of Advanced Sciences, VIT University, Vellore - 632 014. Tamil Nadu, India. . Email. [email protected], [email protected]
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Tel.: +91-416-2202464; Fax: +91-416-224 3092
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Graphical abstract
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Highlights
HAP composite coatings with 0-5% of f-MWCNTs on 316L SS were prepared by ELD method
Contact angle studies showed better hydrophobicity for the 5% HAP/f-MWCNTs coating
Tafel and EIS plots of composite coatings exhibited enhanced corrosion resistance
HAP/f-MWCNTs composite coated 316L SS showed improved Vickers hardness (Hv)
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Abstract
Coatings using functionalized multi-walled carbon nanotubes (f-MWCNTs)/hydroxyapatite (HAP) on 316L
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Stainless Steel by electrodeposition at the parameter of “-1.5 V” for 30 mins with three electrode set-up configuration and optimization of various concentrations of f-MWCNTs from 1 to 5% were done to improve the coating characteristics for future biomedical applications. The obtained coatings were characterized by
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Fourier Transformed-Infra Red spectroscopy (FT-IR) and X-ray diffractometer (XRD) to reveal the phase
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formation in the composites. With various additions of f-MWCNTs, the HAP phase was found to be retained.
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The growth of HAP on f-MWCNTs was analyzed by High-resolution Transmission Electron Microscope (HR-
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TEM) and the morphology of composite was found to be of the needle and flower-like particles. To understand the corrosion resistance effect of the developed HAP/f-MWCNTs composite in SBF, electrochemical
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investigations were carried out using Impedance and Tafel polarization analysis. From the results, it was
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observed that the coatings have enhanced corrosion resistance behavior and bioactivity. In addition, the Vickers Hardness study proved that the prepared HAP/fMWCNTs composite coating was found to have
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improved hardness value of (Hv) 390.2 ± 8.0. Thus, the electrodeposited composite coating on 316L SS substrate can be effectively deployed for biomedical applications.
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Keywords: Electro-deposition,f-MWCNTs, Impedance, Bioactivity, Hardness.
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Introduction 2
Most of the research today is focused on the development of bio ceramic coatings on metallic implants such as Cobalt chromium alloys (Co-Cr), titanium alloys (Ti), stainless steel (316L SS) etc. to improve their corrosion resistance, mechanical strength and bioactivity for orthopedic applications [1-4]. Hydroxyapatite (HAP) with a molecular formula of Ca10(PO4)6(OH)2 is calcium phosphate-based bioceramic material which has properties similar to the human bone and is widely used for coatings on metallic substrates due to its better osteoconductive properties [5-7]. Deposition of HAP on metallic implants can be attained using various
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coating methods such as plasma spraying, a sol-gel method, electrophoretic deposition (EPD) and electrochemical deposition [8-13]. Among all the techniques, plasma spray and EPD were commonly
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employed for HAP coating. Plasma spray method was operated at very high temperatures which may lead to change in phase or even decomposition of HAP and also cannot be used for complex structures. The
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electrophoretic deposition process is conducted at high voltages to charge the particles which may lead to the
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polarization of the metallic substrates to be coated. As an alternative, the electrochemical deposition method
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(an in-situ process) can be done under normal atmospheric conditions at lower operating voltages. And it can
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also be employed for coating of multifaceted structures [14-17]. Even though, the poor adhesion and highly porous film formation by the electrochemical deposition were the major drawbacks that might affect the use
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of coated implants for long-term applications. In order to improve the mechanical strength and corrosion resistivity of the HAP coatings, various materials such as Titania, Zirconia, Silica and Carbon based materials
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have been incorporated to the HAP matrix [18-22]. Carbon nanotubes (CNT) are one of the allotropes of
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carbon which was discovered by Iijima, used in numerous fields due to its extensive properties such as high thermal stability, conductivity, and high mechanical strength. Thus, the mechanical strength of the HAP coatings can be improved by composting it with carbon nanotubes which makes it appropriate for higher load-
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bearing applications. Some recent findings have also been reported on HAP-CNT composite coating with increased mechanical strength using various methods such as electrophoretic deposition, plasma spray etc [2330]. The low solubility of carbon nanotubes leads to agglomeration and makes them difficult to process for different applications. The modification of their sidewalls with suitable functional groups such as -COOH and -OH will enhance their solubility. A negative charge will be created on their surface on functionalization 3
which leads to repulsion between the tubes to be dispersed. Better bonding and improved bioactivity can also be achieved by functionalization [27, 31-33]
In the present study, we have discussed the fabrication of HAP/f-MWCNTs composite coatings through the electro-deposition process. The phase and morphology of the formed composite coatings have been evaluated and their in-vitro corrosion resistance behavior was studied in SBF solution. In vitro bioactivity by SBF immersion analysis and contact angle measurements were carried out for the composite coatings with respect
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to the various concentration of CNT. The schematic representation of this work was shown in the (Fig.S1, ESI) Materials and methods
2.1.
Electrochemical preparation of HAP/f-MWCNTs composite coatings
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2.1. l. Functionalization of MWCNTs
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Prior to the deposition process, the multi-walled carbon nanotubes (MWCNTs, Diameter of 30-50nm, Length
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of 10-30gm) were functionalized and used for the preparation of composite coatings. The pristine MWCNTs
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(0.5g) were suspended in a mixture of concentrated sulphuric acid (H2SO4) and nitric acid (HNO3) (3:1, v/v) and ultra-sonicated for 1 hour. Further, the suspension was refluxed at 100oC for 48 hrs under vigorous stirring.
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After cooling to room temperature, the mixture was filtered using whatmann filter paper. The filtrated solid was then washed thoroughly with an excess of double distilled water until neutral pH was obtained. The
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collected solid was dried in vacuum oven at 70oC for 6 h.
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2.1.2 Substrate preparation
Commercially available surgical grade 316L stainless steel with a dimension of 10 x 10 x 5 mm thickness
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were purchased and used for the studies. The elemental composition (wt%) of the purchased metal substrate as follows: C 0.02, Si 0.15, Mn 1.70, P 0.04, S 0.01, Cr 18, Ni 12, Mo 2.50, N 0.1 and the rest is Fe. The metal specimens were polished sequentially using silicon carbide sheets from grade 120 to 1200 up to mirror finish. Further, the polished specimens were ultrasonically cleaned and degreased using acetone for 15 min. Then the
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samples were again washed with deionized water and stored in a vacuum desiccator to prevent it from oxidation. The polished specimens were further used for the deposition process.
2.1.3 Electrolyte preparation
The electrolyte used for the deposition process contains 0.6M of calcium nitrate tetrahydrate (Ca(NO3)2.4H2O) as calcium source and 0.36M of diammonium hydrogen phosphate ((NH4)2HPO4) as phosphorous source, 1M
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of sodium nitrite (NaNO2) was also added to enhance the ionic activity of the electrolytes and 6% hydrogen peroxide (H2O2) was added to the reaction mixture. The solution pH was maintained between 4.2 and 6 at
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room temperature. The f-MWCNTs were added to the electrolyte solution for the preparation of composite coatings. The various concentrations of f-MWCNTs from 1 to 5% were added to the electrolyte and sonicated
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for an hour to obtain well-dispersed f-MWCNTs.
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2.1.4 Deposition procedure
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The electro-deposition process was carried out using three electrode arrangement, where the polished 316L SS substrate was used as a cathode, platinum wire (Pt) acts as auxiliary electrode and silver/silver chloride
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(Ag/AgCl) as a reference electrode. The deposition of HAP/f-MWCNTs coatings on 316L SS substrate was done by applying the voltage of “-1.5 V” for 30 mins at the temperature of 600C. Further, the coated specimens
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were taken out from electrolytic bath and dried at 800C for overnight. Finally, the dried specimens were
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sintered at 3000C for 2 hours in a vacuum atmosphere and used for further studies [34-37].
2.1.5 HAP/f-MWCNTs composite coatings characterization
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The crystallographic phase analysis and formation of the electrochemically derived HAP/f-MWCNTs coating on 316L SS substrate were analyzed using X-ray diffractometer, Bruker D8 Model, Germany in reflection mode with Cu Kα (λ=1.5405 Å) radiation at a scanning speed of 10/min. The diffraction pattern was recorded in the 2θ range from 100 to 700. And their corresponding functional groups were determined by Fourier Transformed-Infra Red spectra, (Shimadzu-IR affinity spectrometer, Japan) over the frequency range of 4000 5
cm-1 to 400 cm-1 with a spectral resolution of 4 cm-. The morphology and the microstructure of the fabricated HAP and their composite coatings were observed by scanning electron microscopy, using Zeiss EVO 18 research SEM at an operating voltage of 10 - 20 KV equipped with EDAX. The internal morphology of the HAP/f-MWCNTs coating was analysed using High-resolution transmission electron microscope (HR-TEM; Tecnai, G2 20 Twin). A considerable quantity of HAP/f-MWCNTs sample was scraped from the coated substrate and well dispersed in ethanol by means of ultra-sonication for 15 mins. Further, the suspension was
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dropped on copper coated carbon grid and the solvent was evaporated. The surface wettability of the obtained hydroxyapatite composite coatings and uncoated substrate were measured using a camera-assisted contact
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angle instrument (Model Pheonix 300, SEO, Korea). The measurements were conducted by dropping one µl of distilled water onto the surface of prepared metal substrates.
In-vitro corrosion studies
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2.3.
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The corrosion performance of the bare, HAP and HAP/f-MWCNTS coated substrates were studied using
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electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization technique (Tafel). The
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experiments were carried out using the electrochemical workstation (Biologic SA model SP-150, France) with
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three electrode arrangements, wherein the specimen of area with 1x1cm2 was kept as working electrode (WE), Platinum wire as counter electrode (CE), Silver/silver chloride (Ag/AgCl/Satd (KCl)) electrode as reference
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(RE) and Simulated body fluid (SBF) with pH maintained at 7.4 was used as an electrolyte. The electrochemical impedance studies were conducted within a frequency range of 0.1 MHz to 0.01Hz by
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applying AC signal with the small amplitude of 10 mV at the scan speed of 10 points per decade. The polarization studies were carried out by sweeping the electrode potential from -0.300 V to +0.300 V at the
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scan rate of 1 mV per minutes. All the experiments were repeated three times to minimize the error.
2.4.
Apatite formation studies
The bone forming ability studies of the prepared HAP/f-MWCNTS composite coatings on 316L substrates were carried by soaking them in simulated body fluid which mimics the composition and concentration of human blood plasma. The SBF was prepared by following KoKubo et al [38]using different reagents such as 6
NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.224 g), K2HPO4 (0.228), MgCl2.6H2O (0.305 g), CaCl2 (0.278 g), Na2SO4 (0.071 g) and (CH2OH)3 (CNH2).HCl (6.057 g). These mixtures were dissolved in double distilled H2O and the solution pH was adjusted to 7.4 and stored in a refrigerator. The electrochemically derived pure hydroxyapatite and f-MWCNTS embedded HAP coated substrates were immersed in 20 ml of SBF for the period of 7 days at 320C and the SBF solution was refreshed every 24 hours. After the completion of 7 days, the specimens were removed and air dried. Further, the coated substrates were analysed using scanning
Hardness measurement
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2.5.
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electron microscopy to observe the formation of bone-like apatite layer on their surfaces.
Vickers microhardness measurements of the electrolytic deposited pure hydroxyapatite and HAP/f-MWCNTS
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composite substrates were carried using Mutitoyo HM 210B equipped with AVPAK- 20V2.0 software. The
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load of 50g (490.3 mN) was applied with a dwell time of 15s at room temperature. The hardness measurement
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Deposition kinetics
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Results and Discussion
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3.
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for each coated substrates was carried out at least for five times [39].
On electrochemical deposition, when the voltage of -1.5 V was applied to the electrolyte, it leads to the
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production of more hydroxyl ions (OH-) and evolution of hydrogen (H2) gas takes place according to the following equations (1)-(3). The production of OH- ions shifts the pH to of the electrolyte to basic near the
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electrode.
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O2+2H2O + 4e-
NO3- + H2O + 2eH2O + 2e-
4OH-
(1)
2OH- + NO2-
(2)
H2 + 2OH-
(3)
On the other hand, another series of electrochemical reactions also takes place as shown in the chemical equations (4) and (5). These reactions lead to the production of phosphate ions. 7
H2PO4-
HPO4- + H+
(4)
HPO4-
PO43- + H+
(5)
Finally, the produced hydroxyl (OH-) and phosphate ions (PO43-) were precipitated as HAP upon reacting with the calcium ions (Ca2+) in the electrolyte [17, 40, 41]. The following reaction mechanism was presented as
10 Ca2+ + 6 PO43- + 2 OH-
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equation (6)
Ca10(PO4)6(OH)2
(6)
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Additionally, the electrolyte consisting of f-MWCNTs was incorporated into the reaction mixture to form hydroxyapatite/f-MWCNTs coatings. The surface of carbon nanotubes possesses negative charge due to the functionalization by the presence of carboxylic (COO-) and hydroxyl (OH-) ions by surface modification. This
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helps the positively charged Ca2+ to bind with the f-MWCNTs. The hydroxyl (OH-) and phosphate ions
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(PO43-) reacts with calcium ions during the deposition process and leads to the production of hydroxyapatite.
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3.2. Coating characterization 3.2.1. Phase analysis
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The FT-IR spectra of prepared pure HAP and hydroxyapatite/ f-MWCNTs coatings were shown in Fig. la. The observed spectrum confirms the presence of characteristic bands of hydroxyapatite on all the coatings.
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The bands at 1034 cm-1, 914 cm-1, and 553 cm-1 are corresponding to the symmetric and asymmetric stretching
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modes of PO43- groups. The characteristic bands of f-MWCNTs were not observed in any of the spectra. This may be due to the low-level addition of carbon nanotubes into the HAP matrix. The minor shift in the peak position was observed with the addition of f-MWCNTs in the FT-IR spectrum. The bands at 1094-969 and
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triplet bands at 636-567 cm-1 attributed to the presence of HAP in the composite coating. XRD pattern of the electrochemically deposited HAP and its composite coating with f-MWCNTs were shown in the Fig. 1b. The XRD analysis of prepared HAP and HAP/ f-MWCNTs composite coating shows the presence of characteristic peaks at 26o (002), 32.3o (112), 32.9o (300) of HAP which was matching with a standard reference pattern of hydroxyapatite (JCPDS.no.09-0432). Along with HAP, the diffraction peaks of 316L SS substrate was also 8
observed at the peak positions of 43o and 51o, this may be due to porous nature of the HAP coating formed. On increasing the concentration of the f-MWCNTs from 1 to 4% a considerable decrease in the intensity of 316L SS peak has been noted. This is due to the occupation of the f-MWCNTs on the pores of the HAP coatings. Additionally, no other secondary peaks of HAP were observed from the XRD spectrum, which confirms the deposition of pure HAP coatings on the surface of the metallic substrate. The energy dispersive X-ray analysis of the prepared HAP and its composite coatings are shown in (Fig.S2, ESI) the EDAX spectra
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of pure HAP coatings identifies the presence of elements such as Ca, P and O, additionally minor level of Fe corresponds to metal substrate also present which might be due to porous nature of the formed coating. In case
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of, HAP/ f-MWCNTs composite a minor percentage of carbon peaks was additionally detected which confirms the incorporation of carbon nanotubes into the hydroxyapatite matrix. The absence of metallic peak (Fe) was
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also observed in the EDAX spectra of composite coating. This shows that addition of f-MWCNTs leads to the
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formation of less or non-porous coating on the metallic substrate, where the CNTs occupies the voids in
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between the HAP crystals during the electrodeposition process.
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3.2.2. Surface morphology
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The surface morphology of pure HAP and HAP/ f-MWCNTs was analysed using scanning electron microscopy and the results are shown in Fig.2a and 2b. All the coatings prepared by electrolytic deposition
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are found to be in nano phase. The needle, rod, and petal-shaped morphologies were observed for HAP/ f-
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MWCNTs composite coatings. As observed from Fig.2a, the pure HAP coating is found to have fibrous particles on the surface of the implant. With the increase in the percentage of f-MWCNTs, the fibrous nature of the particles is found to be less and completely covered. Additionally at higher magnification, with an
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increase in the percentage of carbon nanotubes into the HAP matrix, the presence of f-MWCNTs and HAP particles were clearly observed. The flower petals morphology of HAP was also found to be retained even with the addition of f-MWCNTs. It was observed that the pure hydroxyapatite coating possesses porous nature, whereas in the composite coatings the f-MWCNTs occupied these pores and form less porous films which can be clearly seen in the higher magnified images of electron microscopy which corroborates with the results 9
from XRD and EDAX analysis. This leads to decrease in the porosity of the formed coatings which relatively increase the mechanical strength and bonding nature of the deposited film. The corrosion resistance behaviour of the coating can also be enhanced by blocking of aggressive ions when exposed to physiological media. Further dense and compact deposit was observed from the cross-sectional image of the 3% f-MWCNTs coating shown in Fig. 3b). The TEM image of the prepared coating (Fig.3a.) shows the better reinforcement of f-MWCNTs into hydroxyapatite matrix and the attachment of HAP rod-like crystals on the f-MWCNTs due
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to surface functionalization are clearly observed by TEM analysis.
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3.2.3. Wettability studies
Surface wettability of the metallic implants is one of the important parameters that control the cell adhesion and growth of the bone tissues on the surface of the implants. If the specimens provide higher wettable
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surfaces, it favors the adhesion of mammalian cells due to higher spreading over the implant surface [42]. The
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wettability of the prepared HAP composite coatings was measured by contact angle method. The contact angle
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studies of the electrochemically derived HAP composite coatings are presented in Fig.4. It is clearly identified
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that the derived HAP and their composite coatings are highly hydrophilic in nature (i.e. lesser angle of the
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water droplet) when compared to that of the uncoated substrate (77.97o± 1.85o). This increased hydrophilic nature of the coated substrates encourages the attachment of cell and their proliferation. Additionally, the
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contact angle was found to increase between 8.14o and 30.08o by the addition of f-MWCNTS from 1 to 5%, which may be due to the decrease in the porous nature of the developed coating due to the occupation of pores
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in between the HAP particles, thereby which restricts the absorption or penetration of water droplet inside
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their matrix. This behavior was in good agreement with the results of SEM analysis.
3.2.4. Vickers Hardness Test
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The Vickers hardness value of the prepared pure HAP and its f-MWCNTs composite coatings were shown in (Fig.4b). Hv value of 273.4 ± 6.68 was observed for the prepared pure hydroxyapatite coated 316 L SS substrates. The HAP/f-MWCNTs composite coated substrates with different concentration of f-MWCNTs i.e. 1%, 3%, and 5% showed increased hardness values of 316.1± 15.7, 357.6 ± 14.8 and 390.2 ± 8.0, respectively. This may be due to the addition of f-MWCNTs into the HAP matrix leads to the formation of high compact
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coatings with higher bonding with metallic substrates.
3.3. Electrochemical studies
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The corrosion resistance behavior of the electrochemically derived HAP and their composite coatings with fMWCNTs were evaluated by electrochemical impedance spectroscopy (EIS) and potentiodynamic
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polarization plots (Tafel).
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3.3.1. Potentiodynamic polarization studies
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The electrochemical property of the coatings prepared through electro-deposition was evaluated by polarization studies and are shown in Fig.5. The following electrokinetic parameters such as corrosion
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potential (Ecorr) and corrosion current density (Icorr) calculated by Tafel extrapolation of cathodic and anodic slopes were presented in Table.1. Initially, the uncoated bare 316L SS substrate shows poor corrosion
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resistance by having a very high Icorr value of 37.497 µA/cm2 and less Ecorr value of -379 mV when compared
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to the coated substrates which express the barrier effect of the HAP composite coatings from the attack of aggressive ions present in the electrolyte. On the other hand, compared to pure hydroxyapatite coating the Ecorr value of all the HAP/ f-MWCNTs composite coatings was shifted towards noble direction. It can also be
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observed that more the addition of f-MWCNTs to HAP more the shift in corrosion potential value to positive direction was inferred. The calculated current density value for pure HAP coating was about 1.427µA/cm2, and it was found to be decreasing with the incorporation of f-MWCNTs into HAP matrix. When compared to the Icorr value of pure HAP coating, the corrosion current density (Icorr) value for the composite coatings were decreased from 1.040 µA/cm2 to 0.053 µA/cm2 respectively with the addition of f-MWCNTs from 1 to 5%. 11
These overall results obtained from polarization studies proved the improved corrosion resistance behaviour of HAP/ f-MWCNTs coatings, further enhanced corrosion protection was gained at higher addition of fMWCNTs. This result confirms the formation of less-porous and impenetrable coating on compositing HAP with carbon nanotubes, where the f-MWCNTs occupy at the voids between the hydroxyapatite particles during the electrochemical deposition process.
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3.3.2. Electrochemical impedance studies
The electrochemical impedance analysis for the HAP coatings with and without f-MWCNTs is shown in Fig.6.
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The spectra was fitted with an equivalent circuit models using Z-fit software and shown in Fig.6d. The equivalent circuit used for uncoated metal substrates were Rs (Q1Rp) ,in which Rs represents the solution resistance , Rp was polarization resistance of the metal substrate and Q1 was electrical double layer capacitance
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of substrate /electrolyte interface. For the HAP/ f-MWCNTs coated substrates, fitting was done using dual-
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layer structure model Rs (Q1 (Rp (Qcoat Rcoatt) , where the Rs represents the electrolyte resistance , Rp was
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polarization resistance of the 316L SS substrates, Q1 was electrical double layer capacitance at the
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substrate/electrolyte interface, Rcoat corresponds to the charge-transfer resistance of the electrochemically
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derived coatings and Qcoat denotes the electrical double layer capacitance at the coating/electrolyte interface. From the values obtained from Nyquist plot (Table.1) it was observed that, the polarization resistance (R p)
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value was increased significantly and capacitance (Q1) was decreased for HAP coated substrates when compared to that of bare substrate which confirms the enhancement of the corrosion resistance behavior by
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preventing the metal substrates from the attack of aggressive ions. The Rp value of 1.02 KΩ cm2 was observed for the un-coated metallic substrate and it was increased from 5.4 to 45.8 KΩ cm2 for the respective HAP
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composite coated substrates. From bode plot (Fig.6b), we observed a shift in the impedance value towards higher magnitude from 103 to 106 for the HAP/ f-MWCNTs composites coated metallic implants. This result accomplishes that electrochemically derived HAP coatings shows effective corrosion resistance performance when compared to that of bare substrates. The shift in the phase angle (Fig.6c.) towards the higher angle at lower frequency region was observed for all the composite coated substrates evident the enhancement of corrosion resistivity in SBF solution. 12
Additionally, the impact of corrosion resistive performance with the addition of f-MWCNTs at different concentrations (1 to 5%) was studied. The pure hydroxyapatite coated 316L SS substrates shows poor (Rcoat) charge transfer resistance when compared to that of composite coatings, this may be due to the porous nature of the formed pure HAP coatings which allows the corrosive ions from the electrolyte to diffuse through their pores and make the prepared film less stable. Alternatively, the additions of f-MWCNTs are found to increase the Rcoat of the coatings with the increase in the concentration of f-MWCNTs from 81.77 KΩ cm2 to 621 KΩ
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cm2. The addition of f-MWCNTs leads to the formation of highly dense layer coating which restricts the diffusion of destructive ions from the electrolyte solution. The impenetrable layer was produced due to the
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occupation of f-MWCNTs into the voids between the HAP particles during the electrochemical deposition process. As the concentration of f-MWCNTs increases the better corrosion protection by the reduction of
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porosity in the coating, the above fact was also supported by the contact angle measurements there it exhibits
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higher hydrophobicity for the 5% HAP/ f-MWCNTs. Electrochemical studies of the prepared coatings
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confirms the corrosion performance in the following order, 5% f-MWCNTs > 4% f-MWCNTs > 3% f-
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MWCNTs > 2% f-MWCNTs > 1% f-MWCNTs > pure HAP coatings > bare 316L SS.
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3.4. In-vitro bioactivity studies
Fig.7 shows the SEM images of the prepared pure HAP and its f-MWCNTs composite coatings after
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immersion in SBF for the duration of 7 days. In the case of, pure HAP coated substrates less precipitation of
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apatite layer was observed on their surfaces for 7days. In f-MWCNTs reinforced hydroxyapatite coated surfaces the better deposition of the apatite layer with spherical bead like formation was observed. The precipitation of carbonated layer on the surface of the implant was encouraged with the addition of f-MWCNTs
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and it was found to increase with an elevation in the concentration of CNT from 1 to 5% as observed from the SEM images. The 5% of f-MWCNT/hydroxyapatite coated substrate showed the presence of well-defined ball-shaped HAP particles, than the other composite derived from less percentage of CNTs. Addition of fMWCNTs in the HAP matrix plays a key role in speeding up the apatite formation process. Porous morphology with interconnected networks of apatite layer was observed on the higher magnification of SEM 13
images (Fig.7e.) which may support the improved cell adhesion, proliferation, and growth of surrounding bone tissues on to their surface.
The mechanism of the carbonated apatite layer formation on the biomaterial surface has been already well proved by many researchers [30, 43]. At first, the Ca2+ ions present in the SBF solution was electrostatically attracted towards the negatively charged surface (OH-) in the HAP thereby forms a positively charged surface. Secondarily, the Ca/P layer was formed due to precipitation of phosphate ions (PO43-) on the cationic calcium
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layer which favorably leads to the formation of the stable bone mineral layer. The OH- group present in the carboxylic functionalized carbon nanotubes acts as an extra nucleating agent for the bio mineralization
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combined with OH- ion present in the HAP. The concentration of OH- was increased with increase in the concentration of f-MWCNTs which provides more sites for the precipitation of more intense apatite layer at a
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faster rate from the physiological solutions. This study proved that the addition of f-MWCNTs leads to
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enhanced mineralization behaviour of apatite layer on the surface.
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4. Conclusions
Hydroxyapatite composite coatings with varied concentration of-MWCNTs (0 to 1%) on 316L SS by
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electrochemical deposition method were fabricated. X-ray diffraction analysis for the prepared coatings confirmed the formation of pure hydroxyapatite even at the high-level addition of carbon nanotubes. The
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needle and lamellar-flower shaped particles with a dense layer of the coating was confirmed by the SEM
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analysis. The incorporation of-MWCNTs into the HAP matrix was confirmed using TEM analysis. Contact angle measurement showed increased hydrophobicity nature for the 5% HAP/f-MWCNTs composite coatings, which may be helpful to increase the corrosion resistive behaviour of the coating in SBF solution. With the
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addition of carboxylic group mediated f-MWCNTs into the HAP matrix, the active site responsible for the formation of carbonated apatite layer was enriched and it may lead to increase in the rate of deposition of bone-like apatite layer on the surface. The potentiodynamic polarization studies of the prepared composite coatings show a noble shift in the Ecorr value with a decrease in the corrosion current density which confirmed the enhanced corrosion resistance properties of the prepared coatings. The above fact was strongly supported 14
by the electrochemical impedance spectroscopic studies; the corrosion resistance behaviour was seemed to be increased with the concentration of f-MWCNTs from 0 to 5% due to the formation of more adherent and nonporous coating. The prepared HAP/f-MWCNTs composite coated 316L SS substrates showed improved hardness strength. Hence, the electrochemically derived HAP/f-MWCNTs composite coatings deposited on
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the 316L SS acts as a promising candidate in the replacement of bone in the biomedical field.
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Acknowledgment
The authors highly acknowledge the DST- Science and Engineering Research Board, (SB-FTCJS-091/2012)
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India for financial support and VIT University, Vellore, India for providing the facilities.
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Fig.1a. FT-IR spectral analysis for the prepared HAP/f-MWCNTs composite coated 316L substrates
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Fig.1b. X-ray diffraction studies for the prepared HAP/f-MWCNTs composite coated 316L substrates a) pure HAP b)HAP/1% f-MWCNTS c) HAP/2% f-MWCNTS d) HAP/3% f-MWCNTS e) HAP/4% fMWCNTS.
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HAP
f-
HAP/ 4% MWCNTS
f-
f-
HAP/ 3% MWCNTS
f-
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HAP/ 2% MWCNTS
HAP/ 1% MWCNTS
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Pure coating
f-
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HAP/ 5% MWCNTS
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Fig.2a. Surface morphological studies for the prepared HAP/f-MWCNTs composite coated substrates using Scanning electron microscopy.
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Fig.2b. Higher magnified SEM images for the prepared HAP/f-MWCNTs composite coated 316L substrates showed the occupation of MWCNTs in the hydroxyapatite matrix.
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Fig.3a. Transmission electron microscopic image of HAP/f-MWCNTs composite at 3%
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Fig.3b. Cross sectional image of HAP@3% f-MWCNTS composite coating on 316L SS substrate.
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Fig.4a. Contact angle measurements for the prepared HAP/f-MWCNTs composite coated 316L substrates.
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Pure HAP coating
HAP@1% f-MWCNTs
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(b) Optical
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Fig.4b. Vickers hardness measurements a) for pure HAP and HAP/f-MWCNTs coatings images of the micro intended surfaces of coated 316L SS substrates.
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Fig.5. TAFEL plots for the prepared HAP/f-MWCNTs composite coated 316L substrates.
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Fig.6a. Nyquist plots for the prepared HAP/f-MWCNTs composite coated 316L substrates
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Fig.6b. Bode impedance curves for the prepared HAP/f-MWCNTs composite coated 316L substrates
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Fig.6c. Phase angle plots for the prepared HAP/f-MWCNTs composite coated 316L substrates
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Fig.6d. Equivalent circuit model used for fitting
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Figure.7. SEM analysis of the coated substrates after 7 days of immersion in SBF a) pure HAP b)HAP/1% f-MWCNTS c) HAP/2% f-MWCNTS d) HAP/3% f-MWCNTS e) HAP/5% f-MWCNTS.
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Table.1. Electro kinetic parameters obtained from electrochemical studies
Rp
2 (Ohms/cm )
(K
ohms/
Q1
R coat
Q coat
Ecorr
Icorr
(Farads)
(K ohms/ 2 cm )
(Farads)
(V)
(µA/cm2)
0.12e-
--
--
0.379
37.497
2 cm )
Bare
21.74
1.02
Pure HAP coated
21.32
5.40
0.50e-
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3
24.27
35.6 e-6
0.082
1.427
81.77
36.6 e-6
0.080
1.040
30.88
4.24
HAP@2% MWCNTs
f-
82.90
11.89
HAP@3% MWCNTs
f-
40.72
HAP MWCNTs
@4%f-
HAP@5% MWCNTs
f-
41.1 e-6
223.6
0.12 e-6
0.021
0.563
19.53
70.5 e-6
283.7
2.40 e-6
0.010
0.418
23.08
45.85
49.1 e-6
480.6
0.11 e-6
0.005
0.211
86.00
28.50
33.7 e-6
621.0
0.56 e-6
0.007
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44.1 e-6
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A
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f-
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4
HAP@1% MWCNTs
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Sample code
Rs
35
0.053
S0927-7765(18)30429-6 https://doi.org/10.1016/j.colsurfb.2018.06.058 COLSUB 9448
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-2-2018 28-5-2018 27-6-2018
Please cite this article as: S. Arul Xavier S, U. V, Electrochemically grown functionalized -Multi-walled carbon nanotubes/hydroxyapatite hybrids on surgical grade 316L SS with enhanced corrosion resistance and bioactivity, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.06.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Electrochemically grown functionalized -Multi-walled carbon nanotubes/hydroxyapatite hybrids on surgical grade 316L SS with enhanced corrosion resistance and bioactivity.
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S. Arul Xavier Stango and U. Vijayalakshmi*
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Department of Chemistry, School of Advanced Sciences, VIT University, Vellore - 632 014. Tamil Nadu, India. . Email. [email protected], [email protected]
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Tel.: +91-416-2202464; Fax: +91-416-224 3092
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Graphical abstract
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Highlights
HAP composite coatings with 0-5% of f-MWCNTs on 316L SS were prepared by ELD method
Contact angle studies showed better hydrophobicity for the 5% HAP/f-MWCNTs coating
Tafel and EIS plots of composite coatings exhibited enhanced corrosion resistance
HAP/f-MWCNTs composite coated 316L SS showed improved Vickers hardness (Hv)
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Abstract
Coatings using functionalized multi-walled carbon nanotubes (f-MWCNTs)/hydroxyapatite (HAP) on 316L
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Stainless Steel by electrodeposition at the parameter of “-1.5 V” for 30 mins with three electrode set-up configuration and optimization of various concentrations of f-MWCNTs from 1 to 5% were done to improve the coating characteristics for future biomedical applications. The obtained coatings were characterized by
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Fourier Transformed-Infra Red spectroscopy (FT-IR) and X-ray diffractometer (XRD) to reveal the phase
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formation in the composites. With various additions of f-MWCNTs, the HAP phase was found to be retained.
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The growth of HAP on f-MWCNTs was analyzed by High-resolution Transmission Electron Microscope (HR-
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TEM) and the morphology of composite was found to be of the needle and flower-like particles. To understand the corrosion resistance effect of the developed HAP/f-MWCNTs composite in SBF, electrochemical
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investigations were carried out using Impedance and Tafel polarization analysis. From the results, it was
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observed that the coatings have enhanced corrosion resistance behavior and bioactivity. In addition, the Vickers Hardness study proved that the prepared HAP/fMWCNTs composite coating was found to have
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improved hardness value of (Hv) 390.2 ± 8.0. Thus, the electrodeposited composite coating on 316L SS substrate can be effectively deployed for biomedical applications.
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Keywords: Electro-deposition,f-MWCNTs, Impedance, Bioactivity, Hardness.
1.
Introduction 2
Most of the research today is focused on the development of bio ceramic coatings on metallic implants such as Cobalt chromium alloys (Co-Cr), titanium alloys (Ti), stainless steel (316L SS) etc. to improve their corrosion resistance, mechanical strength and bioactivity for orthopedic applications [1-4]. Hydroxyapatite (HAP) with a molecular formula of Ca10(PO4)6(OH)2 is calcium phosphate-based bioceramic material which has properties similar to the human bone and is widely used for coatings on metallic substrates due to its better osteoconductive properties [5-7]. Deposition of HAP on metallic implants can be attained using various
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coating methods such as plasma spraying, a sol-gel method, electrophoretic deposition (EPD) and electrochemical deposition [8-13]. Among all the techniques, plasma spray and EPD were commonly
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employed for HAP coating. Plasma spray method was operated at very high temperatures which may lead to change in phase or even decomposition of HAP and also cannot be used for complex structures. The
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electrophoretic deposition process is conducted at high voltages to charge the particles which may lead to the
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polarization of the metallic substrates to be coated. As an alternative, the electrochemical deposition method
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(an in-situ process) can be done under normal atmospheric conditions at lower operating voltages. And it can
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also be employed for coating of multifaceted structures [14-17]. Even though, the poor adhesion and highly porous film formation by the electrochemical deposition were the major drawbacks that might affect the use
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of coated implants for long-term applications. In order to improve the mechanical strength and corrosion resistivity of the HAP coatings, various materials such as Titania, Zirconia, Silica and Carbon based materials
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have been incorporated to the HAP matrix [18-22]. Carbon nanotubes (CNT) are one of the allotropes of
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carbon which was discovered by Iijima, used in numerous fields due to its extensive properties such as high thermal stability, conductivity, and high mechanical strength. Thus, the mechanical strength of the HAP coatings can be improved by composting it with carbon nanotubes which makes it appropriate for higher load-
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bearing applications. Some recent findings have also been reported on HAP-CNT composite coating with increased mechanical strength using various methods such as electrophoretic deposition, plasma spray etc [2330]. The low solubility of carbon nanotubes leads to agglomeration and makes them difficult to process for different applications. The modification of their sidewalls with suitable functional groups such as -COOH and -OH will enhance their solubility. A negative charge will be created on their surface on functionalization 3
which leads to repulsion between the tubes to be dispersed. Better bonding and improved bioactivity can also be achieved by functionalization [27, 31-33]
In the present study, we have discussed the fabrication of HAP/f-MWCNTs composite coatings through the electro-deposition process. The phase and morphology of the formed composite coatings have been evaluated and their in-vitro corrosion resistance behavior was studied in SBF solution. In vitro bioactivity by SBF immersion analysis and contact angle measurements were carried out for the composite coatings with respect
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to the various concentration of CNT. The schematic representation of this work was shown in the (Fig.S1, ESI) Materials and methods
2.1.
Electrochemical preparation of HAP/f-MWCNTs composite coatings
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2.1. l. Functionalization of MWCNTs
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Prior to the deposition process, the multi-walled carbon nanotubes (MWCNTs, Diameter of 30-50nm, Length
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of 10-30gm) were functionalized and used for the preparation of composite coatings. The pristine MWCNTs
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(0.5g) were suspended in a mixture of concentrated sulphuric acid (H2SO4) and nitric acid (HNO3) (3:1, v/v) and ultra-sonicated for 1 hour. Further, the suspension was refluxed at 100oC for 48 hrs under vigorous stirring.
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After cooling to room temperature, the mixture was filtered using whatmann filter paper. The filtrated solid was then washed thoroughly with an excess of double distilled water until neutral pH was obtained. The
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collected solid was dried in vacuum oven at 70oC for 6 h.
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2.1.2 Substrate preparation
Commercially available surgical grade 316L stainless steel with a dimension of 10 x 10 x 5 mm thickness
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were purchased and used for the studies. The elemental composition (wt%) of the purchased metal substrate as follows: C 0.02, Si 0.15, Mn 1.70, P 0.04, S 0.01, Cr 18, Ni 12, Mo 2.50, N 0.1 and the rest is Fe. The metal specimens were polished sequentially using silicon carbide sheets from grade 120 to 1200 up to mirror finish. Further, the polished specimens were ultrasonically cleaned and degreased using acetone for 15 min. Then the
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samples were again washed with deionized water and stored in a vacuum desiccator to prevent it from oxidation. The polished specimens were further used for the deposition process.
2.1.3 Electrolyte preparation
The electrolyte used for the deposition process contains 0.6M of calcium nitrate tetrahydrate (Ca(NO3)2.4H2O) as calcium source and 0.36M of diammonium hydrogen phosphate ((NH4)2HPO4) as phosphorous source, 1M
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of sodium nitrite (NaNO2) was also added to enhance the ionic activity of the electrolytes and 6% hydrogen peroxide (H2O2) was added to the reaction mixture. The solution pH was maintained between 4.2 and 6 at
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room temperature. The f-MWCNTs were added to the electrolyte solution for the preparation of composite coatings. The various concentrations of f-MWCNTs from 1 to 5% were added to the electrolyte and sonicated
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for an hour to obtain well-dispersed f-MWCNTs.
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2.1.4 Deposition procedure
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The electro-deposition process was carried out using three electrode arrangement, where the polished 316L SS substrate was used as a cathode, platinum wire (Pt) acts as auxiliary electrode and silver/silver chloride
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(Ag/AgCl) as a reference electrode. The deposition of HAP/f-MWCNTs coatings on 316L SS substrate was done by applying the voltage of “-1.5 V” for 30 mins at the temperature of 600C. Further, the coated specimens
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were taken out from electrolytic bath and dried at 800C for overnight. Finally, the dried specimens were
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sintered at 3000C for 2 hours in a vacuum atmosphere and used for further studies [34-37].
2.1.5 HAP/f-MWCNTs composite coatings characterization
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The crystallographic phase analysis and formation of the electrochemically derived HAP/f-MWCNTs coating on 316L SS substrate were analyzed using X-ray diffractometer, Bruker D8 Model, Germany in reflection mode with Cu Kα (λ=1.5405 Å) radiation at a scanning speed of 10/min. The diffraction pattern was recorded in the 2θ range from 100 to 700. And their corresponding functional groups were determined by Fourier Transformed-Infra Red spectra, (Shimadzu-IR affinity spectrometer, Japan) over the frequency range of 4000 5
cm-1 to 400 cm-1 with a spectral resolution of 4 cm-. The morphology and the microstructure of the fabricated HAP and their composite coatings were observed by scanning electron microscopy, using Zeiss EVO 18 research SEM at an operating voltage of 10 - 20 KV equipped with EDAX. The internal morphology of the HAP/f-MWCNTs coating was analysed using High-resolution transmission electron microscope (HR-TEM; Tecnai, G2 20 Twin). A considerable quantity of HAP/f-MWCNTs sample was scraped from the coated substrate and well dispersed in ethanol by means of ultra-sonication for 15 mins. Further, the suspension was
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dropped on copper coated carbon grid and the solvent was evaporated. The surface wettability of the obtained hydroxyapatite composite coatings and uncoated substrate were measured using a camera-assisted contact
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angle instrument (Model Pheonix 300, SEO, Korea). The measurements were conducted by dropping one µl of distilled water onto the surface of prepared metal substrates.
In-vitro corrosion studies
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2.3.
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The corrosion performance of the bare, HAP and HAP/f-MWCNTS coated substrates were studied using
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electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization technique (Tafel). The
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experiments were carried out using the electrochemical workstation (Biologic SA model SP-150, France) with
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three electrode arrangements, wherein the specimen of area with 1x1cm2 was kept as working electrode (WE), Platinum wire as counter electrode (CE), Silver/silver chloride (Ag/AgCl/Satd (KCl)) electrode as reference
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(RE) and Simulated body fluid (SBF) with pH maintained at 7.4 was used as an electrolyte. The electrochemical impedance studies were conducted within a frequency range of 0.1 MHz to 0.01Hz by
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applying AC signal with the small amplitude of 10 mV at the scan speed of 10 points per decade. The polarization studies were carried out by sweeping the electrode potential from -0.300 V to +0.300 V at the
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scan rate of 1 mV per minutes. All the experiments were repeated three times to minimize the error.
2.4.
Apatite formation studies
The bone forming ability studies of the prepared HAP/f-MWCNTS composite coatings on 316L substrates were carried by soaking them in simulated body fluid which mimics the composition and concentration of human blood plasma. The SBF was prepared by following KoKubo et al [38]using different reagents such as 6
NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.224 g), K2HPO4 (0.228), MgCl2.6H2O (0.305 g), CaCl2 (0.278 g), Na2SO4 (0.071 g) and (CH2OH)3 (CNH2).HCl (6.057 g). These mixtures were dissolved in double distilled H2O and the solution pH was adjusted to 7.4 and stored in a refrigerator. The electrochemically derived pure hydroxyapatite and f-MWCNTS embedded HAP coated substrates were immersed in 20 ml of SBF for the period of 7 days at 320C and the SBF solution was refreshed every 24 hours. After the completion of 7 days, the specimens were removed and air dried. Further, the coated substrates were analysed using scanning
Hardness measurement
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2.5.
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electron microscopy to observe the formation of bone-like apatite layer on their surfaces.
Vickers microhardness measurements of the electrolytic deposited pure hydroxyapatite and HAP/f-MWCNTS
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composite substrates were carried using Mutitoyo HM 210B equipped with AVPAK- 20V2.0 software. The
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load of 50g (490.3 mN) was applied with a dwell time of 15s at room temperature. The hardness measurement
3.1.
Deposition kinetics
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Results and Discussion
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3.
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for each coated substrates was carried out at least for five times [39].
On electrochemical deposition, when the voltage of -1.5 V was applied to the electrolyte, it leads to the
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production of more hydroxyl ions (OH-) and evolution of hydrogen (H2) gas takes place according to the following equations (1)-(3). The production of OH- ions shifts the pH to of the electrolyte to basic near the
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electrode.
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O2+2H2O + 4e-
NO3- + H2O + 2eH2O + 2e-
4OH-
(1)
2OH- + NO2-
(2)
H2 + 2OH-
(3)
On the other hand, another series of electrochemical reactions also takes place as shown in the chemical equations (4) and (5). These reactions lead to the production of phosphate ions. 7
H2PO4-
HPO4- + H+
(4)
HPO4-
PO43- + H+
(5)
Finally, the produced hydroxyl (OH-) and phosphate ions (PO43-) were precipitated as HAP upon reacting with the calcium ions (Ca2+) in the electrolyte [17, 40, 41]. The following reaction mechanism was presented as
10 Ca2+ + 6 PO43- + 2 OH-
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equation (6)
Ca10(PO4)6(OH)2
(6)
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Additionally, the electrolyte consisting of f-MWCNTs was incorporated into the reaction mixture to form hydroxyapatite/f-MWCNTs coatings. The surface of carbon nanotubes possesses negative charge due to the functionalization by the presence of carboxylic (COO-) and hydroxyl (OH-) ions by surface modification. This
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helps the positively charged Ca2+ to bind with the f-MWCNTs. The hydroxyl (OH-) and phosphate ions
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(PO43-) reacts with calcium ions during the deposition process and leads to the production of hydroxyapatite.
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3.2. Coating characterization 3.2.1. Phase analysis
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The FT-IR spectra of prepared pure HAP and hydroxyapatite/ f-MWCNTs coatings were shown in Fig. la. The observed spectrum confirms the presence of characteristic bands of hydroxyapatite on all the coatings.
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The bands at 1034 cm-1, 914 cm-1, and 553 cm-1 are corresponding to the symmetric and asymmetric stretching
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modes of PO43- groups. The characteristic bands of f-MWCNTs were not observed in any of the spectra. This may be due to the low-level addition of carbon nanotubes into the HAP matrix. The minor shift in the peak position was observed with the addition of f-MWCNTs in the FT-IR spectrum. The bands at 1094-969 and
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triplet bands at 636-567 cm-1 attributed to the presence of HAP in the composite coating. XRD pattern of the electrochemically deposited HAP and its composite coating with f-MWCNTs were shown in the Fig. 1b. The XRD analysis of prepared HAP and HAP/ f-MWCNTs composite coating shows the presence of characteristic peaks at 26o (002), 32.3o (112), 32.9o (300) of HAP which was matching with a standard reference pattern of hydroxyapatite (JCPDS.no.09-0432). Along with HAP, the diffraction peaks of 316L SS substrate was also 8
observed at the peak positions of 43o and 51o, this may be due to porous nature of the HAP coating formed. On increasing the concentration of the f-MWCNTs from 1 to 4% a considerable decrease in the intensity of 316L SS peak has been noted. This is due to the occupation of the f-MWCNTs on the pores of the HAP coatings. Additionally, no other secondary peaks of HAP were observed from the XRD spectrum, which confirms the deposition of pure HAP coatings on the surface of the metallic substrate. The energy dispersive X-ray analysis of the prepared HAP and its composite coatings are shown in (Fig.S2, ESI) the EDAX spectra
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of pure HAP coatings identifies the presence of elements such as Ca, P and O, additionally minor level of Fe corresponds to metal substrate also present which might be due to porous nature of the formed coating. In case
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of, HAP/ f-MWCNTs composite a minor percentage of carbon peaks was additionally detected which confirms the incorporation of carbon nanotubes into the hydroxyapatite matrix. The absence of metallic peak (Fe) was
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also observed in the EDAX spectra of composite coating. This shows that addition of f-MWCNTs leads to the
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formation of less or non-porous coating on the metallic substrate, where the CNTs occupies the voids in
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between the HAP crystals during the electrodeposition process.
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3.2.2. Surface morphology
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The surface morphology of pure HAP and HAP/ f-MWCNTs was analysed using scanning electron microscopy and the results are shown in Fig.2a and 2b. All the coatings prepared by electrolytic deposition
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are found to be in nano phase. The needle, rod, and petal-shaped morphologies were observed for HAP/ f-
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MWCNTs composite coatings. As observed from Fig.2a, the pure HAP coating is found to have fibrous particles on the surface of the implant. With the increase in the percentage of f-MWCNTs, the fibrous nature of the particles is found to be less and completely covered. Additionally at higher magnification, with an
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increase in the percentage of carbon nanotubes into the HAP matrix, the presence of f-MWCNTs and HAP particles were clearly observed. The flower petals morphology of HAP was also found to be retained even with the addition of f-MWCNTs. It was observed that the pure hydroxyapatite coating possesses porous nature, whereas in the composite coatings the f-MWCNTs occupied these pores and form less porous films which can be clearly seen in the higher magnified images of electron microscopy which corroborates with the results 9
from XRD and EDAX analysis. This leads to decrease in the porosity of the formed coatings which relatively increase the mechanical strength and bonding nature of the deposited film. The corrosion resistance behaviour of the coating can also be enhanced by blocking of aggressive ions when exposed to physiological media. Further dense and compact deposit was observed from the cross-sectional image of the 3% f-MWCNTs coating shown in Fig. 3b). The TEM image of the prepared coating (Fig.3a.) shows the better reinforcement of f-MWCNTs into hydroxyapatite matrix and the attachment of HAP rod-like crystals on the f-MWCNTs due
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to surface functionalization are clearly observed by TEM analysis.
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3.2.3. Wettability studies
Surface wettability of the metallic implants is one of the important parameters that control the cell adhesion and growth of the bone tissues on the surface of the implants. If the specimens provide higher wettable
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surfaces, it favors the adhesion of mammalian cells due to higher spreading over the implant surface [42]. The
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wettability of the prepared HAP composite coatings was measured by contact angle method. The contact angle
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studies of the electrochemically derived HAP composite coatings are presented in Fig.4. It is clearly identified
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that the derived HAP and their composite coatings are highly hydrophilic in nature (i.e. lesser angle of the
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water droplet) when compared to that of the uncoated substrate (77.97o± 1.85o). This increased hydrophilic nature of the coated substrates encourages the attachment of cell and their proliferation. Additionally, the
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contact angle was found to increase between 8.14o and 30.08o by the addition of f-MWCNTS from 1 to 5%, which may be due to the decrease in the porous nature of the developed coating due to the occupation of pores
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in between the HAP particles, thereby which restricts the absorption or penetration of water droplet inside
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their matrix. This behavior was in good agreement with the results of SEM analysis.
3.2.4. Vickers Hardness Test
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The Vickers hardness value of the prepared pure HAP and its f-MWCNTs composite coatings were shown in (Fig.4b). Hv value of 273.4 ± 6.68 was observed for the prepared pure hydroxyapatite coated 316 L SS substrates. The HAP/f-MWCNTs composite coated substrates with different concentration of f-MWCNTs i.e. 1%, 3%, and 5% showed increased hardness values of 316.1± 15.7, 357.6 ± 14.8 and 390.2 ± 8.0, respectively. This may be due to the addition of f-MWCNTs into the HAP matrix leads to the formation of high compact
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coatings with higher bonding with metallic substrates.
3.3. Electrochemical studies
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The corrosion resistance behavior of the electrochemically derived HAP and their composite coatings with fMWCNTs were evaluated by electrochemical impedance spectroscopy (EIS) and potentiodynamic
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polarization plots (Tafel).
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3.3.1. Potentiodynamic polarization studies
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The electrochemical property of the coatings prepared through electro-deposition was evaluated by polarization studies and are shown in Fig.5. The following electrokinetic parameters such as corrosion
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potential (Ecorr) and corrosion current density (Icorr) calculated by Tafel extrapolation of cathodic and anodic slopes were presented in Table.1. Initially, the uncoated bare 316L SS substrate shows poor corrosion
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resistance by having a very high Icorr value of 37.497 µA/cm2 and less Ecorr value of -379 mV when compared
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to the coated substrates which express the barrier effect of the HAP composite coatings from the attack of aggressive ions present in the electrolyte. On the other hand, compared to pure hydroxyapatite coating the Ecorr value of all the HAP/ f-MWCNTs composite coatings was shifted towards noble direction. It can also be
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observed that more the addition of f-MWCNTs to HAP more the shift in corrosion potential value to positive direction was inferred. The calculated current density value for pure HAP coating was about 1.427µA/cm2, and it was found to be decreasing with the incorporation of f-MWCNTs into HAP matrix. When compared to the Icorr value of pure HAP coating, the corrosion current density (Icorr) value for the composite coatings were decreased from 1.040 µA/cm2 to 0.053 µA/cm2 respectively with the addition of f-MWCNTs from 1 to 5%. 11
These overall results obtained from polarization studies proved the improved corrosion resistance behaviour of HAP/ f-MWCNTs coatings, further enhanced corrosion protection was gained at higher addition of fMWCNTs. This result confirms the formation of less-porous and impenetrable coating on compositing HAP with carbon nanotubes, where the f-MWCNTs occupy at the voids between the hydroxyapatite particles during the electrochemical deposition process.
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3.3.2. Electrochemical impedance studies
The electrochemical impedance analysis for the HAP coatings with and without f-MWCNTs is shown in Fig.6.
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The spectra was fitted with an equivalent circuit models using Z-fit software and shown in Fig.6d. The equivalent circuit used for uncoated metal substrates were Rs (Q1Rp) ,in which Rs represents the solution resistance , Rp was polarization resistance of the metal substrate and Q1 was electrical double layer capacitance
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of substrate /electrolyte interface. For the HAP/ f-MWCNTs coated substrates, fitting was done using dual-
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layer structure model Rs (Q1 (Rp (Qcoat Rcoatt) , where the Rs represents the electrolyte resistance , Rp was
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polarization resistance of the 316L SS substrates, Q1 was electrical double layer capacitance at the
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substrate/electrolyte interface, Rcoat corresponds to the charge-transfer resistance of the electrochemically
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derived coatings and Qcoat denotes the electrical double layer capacitance at the coating/electrolyte interface. From the values obtained from Nyquist plot (Table.1) it was observed that, the polarization resistance (R p)
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value was increased significantly and capacitance (Q1) was decreased for HAP coated substrates when compared to that of bare substrate which confirms the enhancement of the corrosion resistance behavior by
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preventing the metal substrates from the attack of aggressive ions. The Rp value of 1.02 KΩ cm2 was observed for the un-coated metallic substrate and it was increased from 5.4 to 45.8 KΩ cm2 for the respective HAP
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composite coated substrates. From bode plot (Fig.6b), we observed a shift in the impedance value towards higher magnitude from 103 to 106 for the HAP/ f-MWCNTs composites coated metallic implants. This result accomplishes that electrochemically derived HAP coatings shows effective corrosion resistance performance when compared to that of bare substrates. The shift in the phase angle (Fig.6c.) towards the higher angle at lower frequency region was observed for all the composite coated substrates evident the enhancement of corrosion resistivity in SBF solution. 12
Additionally, the impact of corrosion resistive performance with the addition of f-MWCNTs at different concentrations (1 to 5%) was studied. The pure hydroxyapatite coated 316L SS substrates shows poor (Rcoat) charge transfer resistance when compared to that of composite coatings, this may be due to the porous nature of the formed pure HAP coatings which allows the corrosive ions from the electrolyte to diffuse through their pores and make the prepared film less stable. Alternatively, the additions of f-MWCNTs are found to increase the Rcoat of the coatings with the increase in the concentration of f-MWCNTs from 81.77 KΩ cm2 to 621 KΩ
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cm2. The addition of f-MWCNTs leads to the formation of highly dense layer coating which restricts the diffusion of destructive ions from the electrolyte solution. The impenetrable layer was produced due to the
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occupation of f-MWCNTs into the voids between the HAP particles during the electrochemical deposition process. As the concentration of f-MWCNTs increases the better corrosion protection by the reduction of
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porosity in the coating, the above fact was also supported by the contact angle measurements there it exhibits
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higher hydrophobicity for the 5% HAP/ f-MWCNTs. Electrochemical studies of the prepared coatings
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confirms the corrosion performance in the following order, 5% f-MWCNTs > 4% f-MWCNTs > 3% f-
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MWCNTs > 2% f-MWCNTs > 1% f-MWCNTs > pure HAP coatings > bare 316L SS.
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3.4. In-vitro bioactivity studies
Fig.7 shows the SEM images of the prepared pure HAP and its f-MWCNTs composite coatings after
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immersion in SBF for the duration of 7 days. In the case of, pure HAP coated substrates less precipitation of
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apatite layer was observed on their surfaces for 7days. In f-MWCNTs reinforced hydroxyapatite coated surfaces the better deposition of the apatite layer with spherical bead like formation was observed. The precipitation of carbonated layer on the surface of the implant was encouraged with the addition of f-MWCNTs
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and it was found to increase with an elevation in the concentration of CNT from 1 to 5% as observed from the SEM images. The 5% of f-MWCNT/hydroxyapatite coated substrate showed the presence of well-defined ball-shaped HAP particles, than the other composite derived from less percentage of CNTs. Addition of fMWCNTs in the HAP matrix plays a key role in speeding up the apatite formation process. Porous morphology with interconnected networks of apatite layer was observed on the higher magnification of SEM 13
images (Fig.7e.) which may support the improved cell adhesion, proliferation, and growth of surrounding bone tissues on to their surface.
The mechanism of the carbonated apatite layer formation on the biomaterial surface has been already well proved by many researchers [30, 43]. At first, the Ca2+ ions present in the SBF solution was electrostatically attracted towards the negatively charged surface (OH-) in the HAP thereby forms a positively charged surface. Secondarily, the Ca/P layer was formed due to precipitation of phosphate ions (PO43-) on the cationic calcium
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layer which favorably leads to the formation of the stable bone mineral layer. The OH- group present in the carboxylic functionalized carbon nanotubes acts as an extra nucleating agent for the bio mineralization
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combined with OH- ion present in the HAP. The concentration of OH- was increased with increase in the concentration of f-MWCNTs which provides more sites for the precipitation of more intense apatite layer at a
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faster rate from the physiological solutions. This study proved that the addition of f-MWCNTs leads to
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enhanced mineralization behaviour of apatite layer on the surface.
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4. Conclusions
Hydroxyapatite composite coatings with varied concentration of-MWCNTs (0 to 1%) on 316L SS by
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electrochemical deposition method were fabricated. X-ray diffraction analysis for the prepared coatings confirmed the formation of pure hydroxyapatite even at the high-level addition of carbon nanotubes. The
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needle and lamellar-flower shaped particles with a dense layer of the coating was confirmed by the SEM
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analysis. The incorporation of-MWCNTs into the HAP matrix was confirmed using TEM analysis. Contact angle measurement showed increased hydrophobicity nature for the 5% HAP/f-MWCNTs composite coatings, which may be helpful to increase the corrosion resistive behaviour of the coating in SBF solution. With the
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addition of carboxylic group mediated f-MWCNTs into the HAP matrix, the active site responsible for the formation of carbonated apatite layer was enriched and it may lead to increase in the rate of deposition of bone-like apatite layer on the surface. The potentiodynamic polarization studies of the prepared composite coatings show a noble shift in the Ecorr value with a decrease in the corrosion current density which confirmed the enhanced corrosion resistance properties of the prepared coatings. The above fact was strongly supported 14
by the electrochemical impedance spectroscopic studies; the corrosion resistance behaviour was seemed to be increased with the concentration of f-MWCNTs from 0 to 5% due to the formation of more adherent and nonporous coating. The prepared HAP/f-MWCNTs composite coated 316L SS substrates showed improved hardness strength. Hence, the electrochemically derived HAP/f-MWCNTs composite coatings deposited on
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the 316L SS acts as a promising candidate in the replacement of bone in the biomedical field.
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Acknowledgment
The authors highly acknowledge the DST- Science and Engineering Research Board, (SB-FTCJS-091/2012)
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India for financial support and VIT University, Vellore, India for providing the facilities.
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SC R
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ED
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PT
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CC E
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A
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20
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A
CC E
Fig.1a. FT-IR spectral analysis for the prepared HAP/f-MWCNTs composite coated 316L substrates
21
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A
CC E
Fig.1b. X-ray diffraction studies for the prepared HAP/f-MWCNTs composite coated 316L substrates a) pure HAP b)HAP/1% f-MWCNTS c) HAP/2% f-MWCNTS d) HAP/3% f-MWCNTS e) HAP/4% fMWCNTS.
22
HAP
f-
HAP/ 4% MWCNTS
f-
f-
HAP/ 3% MWCNTS
f-
A
N
U
SC R
HAP/ 2% MWCNTS
HAP/ 1% MWCNTS
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Pure coating
f-
CC E
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M
HAP/ 5% MWCNTS
A
Fig.2a. Surface morphological studies for the prepared HAP/f-MWCNTs composite coated substrates using Scanning electron microscopy.
23
IP T SC R U N A M ED PT CC E
A
Fig.2b. Higher magnified SEM images for the prepared HAP/f-MWCNTs composite coated 316L substrates showed the occupation of MWCNTs in the hydroxyapatite matrix.
24
IP T SC R U N A M ED
A
CC E
PT
Fig.3a. Transmission electron microscopic image of HAP/f-MWCNTs composite at 3%
25
IP T SC R
A
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PT
ED
M
A
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Fig.3b. Cross sectional image of HAP@3% f-MWCNTS composite coating on 316L SS substrate.
26
IP T SC R U N A M ED PT CC E
A
Fig.4a. Contact angle measurements for the prepared HAP/f-MWCNTs composite coated 316L substrates.
27
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M
A
N
U
SC R
A
A
CC E
PT
B
Pure HAP coating
HAP@1% f-MWCNTs
28
(b) Optical
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ED
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A
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U
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Fig.4b. Vickers hardness measurements a) for pure HAP and HAP/f-MWCNTs coatings images of the micro intended surfaces of coated 316L SS substrates.
A
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Fig.5. TAFEL plots for the prepared HAP/f-MWCNTs composite coated 316L substrates.
29
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A
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Fig.6a. Nyquist plots for the prepared HAP/f-MWCNTs composite coated 316L substrates
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A
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Fig.6b. Bode impedance curves for the prepared HAP/f-MWCNTs composite coated 316L substrates
31
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A
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Fig.6c. Phase angle plots for the prepared HAP/f-MWCNTs composite coated 316L substrates
32
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A
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A
N
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Fig.6d. Equivalent circuit model used for fitting
33
C
D
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B
A
N
U
SC R
A
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PT
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M
E
A
Figure.7. SEM analysis of the coated substrates after 7 days of immersion in SBF a) pure HAP b)HAP/1% f-MWCNTS c) HAP/2% f-MWCNTS d) HAP/3% f-MWCNTS e) HAP/5% f-MWCNTS.
34
Table.1. Electro kinetic parameters obtained from electrochemical studies
Rp
2 (Ohms/cm )
(K
ohms/
Q1
R coat
Q coat
Ecorr
Icorr
(Farads)
(K ohms/ 2 cm )
(Farads)
(V)
(µA/cm2)
0.12e-
--
--
0.379
37.497
2 cm )
Bare
21.74
1.02
Pure HAP coated
21.32
5.40
0.50e-
SC R
3
24.27
35.6 e-6
0.082
1.427
81.77
36.6 e-6
0.080
1.040
30.88
4.24
HAP@2% MWCNTs
f-
82.90
11.89
HAP@3% MWCNTs
f-
40.72
HAP MWCNTs
@4%f-
HAP@5% MWCNTs
f-
41.1 e-6
223.6
0.12 e-6
0.021
0.563
19.53
70.5 e-6
283.7
2.40 e-6
0.010
0.418
23.08
45.85
49.1 e-6
480.6
0.11 e-6
0.005
0.211
86.00
28.50
33.7 e-6
621.0
0.56 e-6
0.007
ED
M
44.1 e-6
PT
CC E
A
A
N
f-
U
4
HAP@1% MWCNTs
IP T
Sample code
Rs
35
0.053