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The effect of lignin on the structure and characteristics of composite coatings electrodeposited on titanium
Progress in Organic Coatings 75 (2012) 275–283
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Progress in Organic Coatings journal homepage: www.elsevier.com/locate/porgcoat
Review
The effect of lignin on the structure and characteristics of composite coatings electrodeposited on titanium Sanja Erakovic´ a , Djordje Veljovic´ a , Papa N. Diouf b , Tatjana Stevanovic´ c , Miodrag Mitric´ d , ´ ´ Djordje Janackovi c´ a , Ivana Z. Matic´ e , Zorica D. Juranic´ e , Vesna Miˇskovic-Stankovi c´ a,∗ a
Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia Service de recherche et d’expertise en transformation des produits forestiers, 25 rue Armand-Sinclair, porte 5, Amqui, Canada c Département des sciences du bois et de la forêt, Université Laval, 2425 rue de la Terrasse, Québec, Canada d Vinˇca Institute of Nuclear Sciences, University of Belgrade, Mike Petrovi´ca Alasa 12–14, 11000 Belgrade, Serbia e Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 3 July 2012 Accepted 13 July 2012 Available online 4 August 2012 Keywords: Electrophoretic deposition Composite coating Organosolv lignin Hydroxyapatite Titanium Cytotoxicity
a b s t r a c t Composite coatings based on lignin, obtained by electrophoretic deposition (EPD) on titanium, were investigated. The aim of this work was to produce hydroxyapatite/lignin (HAP/Lig) coatings on titanium and to investigate the effect of the lignin concentration on microstructure, morphology, phase composition, thermal behavior and cytotoxicity of the HAP/Lig coatings. An organosolv lignin was used for the production of the composite coatings studied in this research. The properties of HAP/Lig coatings were characterized using X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) and X-ray photoelectron spectroscopy (XPS), as well as the MTT test of cytotoxicity. The results showed that higher lignin concentrations protected the HAP lattice during sintering, thereby improving the stability of the HAP/Lig coatings. The cell survival of peripheral blood mononuclear cells (PBMC) after proliferation indicates that the HAP/Lig coating with 1 wt% Lig electrodeposited on titanium was non-toxic with significant promise as a potential bone-repair material. © 2012 Elsevier B.V. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Alcell lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Nano-sized HAP powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Electrophoretic deposition of composite HAP/Lig coatings on titanium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Characterization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. XRD analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. SEM analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. TG/DTG analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. ATR-FT-IR analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. XPS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +381 11 3303 737; fax: +381 11 3370 387. ´ ´ E-mail address: [email protected] (V. Miˇskovic-Stankovi c). 0300-9440/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.porgcoat.2012.07.005
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1. Introduction Electrophoretic deposition (EPD) is gaining increasing attention as a powerful method for the formation of both uniform thin and thick films on substrates of complex geometry [1–12]. This method is worldwide used for the deposition of organic protective coatings [6–9], ceramic coatings [10–12], bioceramic coatings [2–4,13] and composite coatings [5,14]. In the past few decades, EPD has become an attractive technique for the formation of bioactive ceramic coatings, such as hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HAP), on metallic implants [2]. Titanium and some of its alloys are most often the material of choice for metallic implants due to their attributes of strength, stiffness, toughness, impact resistance and corrosion resistance [15–17]. Titanium has proven itself to be potentially a very suitable material for orthopedic and dental implants, due to its excellent corrosion resistance and biocompatibility. Calcium phosphate coated on metal implants provides the necessary porosity for bone in-growths, therefore HAP is often used as a coating material [17]. Hence, a good combination of the biocompatibility of hydroxyapatite and the excellent mechanical properties of metals is considered a promising approach to fabricate more suitable bone implants. However, the application of pure electrodeposited HAP coatings on metal implants has been limited due to problems such as low adhesion. In order to improve the mechanical properties, adhesion and the brittleness of HAP coatings, composite HAP coatings containing biopolymers have become of great interest [18–20]. As complex natural polymer networks composed primarily of phenolic moieties, lignins have a wide variety of chemical bonds [21,22]. Among the functional groups present in lignin, the most reactive chemical sites are phenolic hydroxyl groups [23]. Other major chemical functional groups in lignins include methoxyl, carbonyl and carboxyl groups, depending on the plant origin and the applied pulping processes [24]. Organosolv lignins are being examined because they show significantly better solubility and thermal properties than sulfite or kraft lignins [25]. The organosolv processes are convenient because of the greater simplicity of the chemical recovery system, since only the solvent has to be recovered by rectification of the black liquors [23]. In contrast to Kraft and sulfite lignin, Alcell (organosolv) lignin derives from a process using ethanol as the only pulping chemical and consists of low molecular weight phenol fragments with enhanced hydrophobicity [22]. Hence, this organosolv lignin, in its purified form, possesses a chemical structure different to that of native lignin. The incorporation of lignin is interesting in medical applications because of its thermal stability and biocompatibility in different materials [26–28]. In this work, the influence of the lignin concentration on the microstructure, morphology, phase composition, thermal behavior and cytotoxicity of composite HAP/Lig coatings electrodeposited on titanium was investigated.
2. Experimental 2.1. Materials 2.1.1. Alcell lignin The lignin powder used without further purification was Alcell lignin from Repap Enterprises Inc. (Stamford, CT) that was extracted from a mixture of hardwoods (maple, birch and poplar) by an organosolv process using aqueous ethanol. The characteristics of the employed AlcellTM lignin were reported in a previous paper [21].
2.1.2. Nano-sized HAP powder A nano-sized HAP powder was obtained using a modified chemical precipitation method, by the reaction of calcium oxide (obtained by calcination of CaCO3 for 5 h at 1000 ◦ C in air) with phosphoric acid, as described in a previous paper [21]. Small portions of the resulting calcium oxide were mixed and stirred with distilled water for 10 min. A calculated amount of phosphoric acid was added dropwise to the calcium hydroxide suspension until the pH reached the value 7.4–7.6. The obtained suspension was heated to 94 ± 1 ◦ C for 30 min and stirred for a further 30 min. After sedimentation, the upper clear solution layer was decanted. The suspension was then spray dried at 120 ± 5 ◦ C into a granulated powder. It was determined by TEM analysis that the mean particle size of the rod-shaped HAP powder was between 50 and 100 nm [29]. 2.2. Electrophoretic deposition of composite HAP/Lig coatings on titanium Electrophoretic deposition was performed from ethanol suspensions containing 1.0 g of nano-sized HAP powder and 0.5–10 wt% of lignin powder in 100 ml of absolute ethanol after ultrasonic treatment for 15 min to obtain homogeneous and stable suspensions. To increase the stability of the suspensions, HCl was added until a pH value of 2.00 was attained. Prior to electrodeposition, the mixtures were ultrasonicated for 30 min to obtain a homogeneous suspension of the particles. A three-electrode cell arrangement was used for the cathodic electrodeposition. The working electrode used as a substrate for the deposition of HAP and HAP/Lig coatings was a titanium plate (65 mm × 5 mm × 0.25 mm, Aldrich, purity 99.7%). The Ti surface was pretreated by polishing with grit emery paper, followed by wet polishing with 0.3 m alumina. After polishing, the titanium plates were degreased with acetone and then with ethanol for 15 min in an ultrasonic bath. The counter electrodes were two platinum panels, placed parallel to the Ti electrode at a distance of 1.5 cm. The HAP and HAP/Lig coatings were obtained on titanium from ethanol HAP and HAP/Lig suspensions using the constant voltage method at a deposition voltage of 60 V for a deposition time of 45 s, at room temperature. The deposited HAP and HAP/Lig coatings were air dried at room temperature and then sintered at 900 ◦ C for 30 min, with the initial heating rate of 16 ◦ C/min, under an argon atmosphere. Before sintering, deoxidization the coatings were deoxidized at 200 ◦ C under an argon atmosphere for 45 min. 2.3. Characterization techniques The phase composition of HAP/Lig coatings was investigated by X-ray diffraction (XRD) analysis using a Philips PW 1051 Powder ˚ The Diffractometer with Ni filtered CuK˛ radiation ( = 1.5418 A). diffraction intensity was measured using the scan-step technique in the 2 range 8–80◦ with a scanning step width of 0.05◦ and exposition time of 50 s per step. The phase analysis was realized using the PDF-2 data base with a commercially available computer program, EVA V.9.0. The average crystallite domain size (Dp ) was calculated from the half height width (ˇ1/2 ) of the XRD reflection of the (0 0 2) plane (at 2 = 25.8◦ ), using the Scherer equation (1): Dp =
K ˇ1/2 cos
(1)
where is the wavelength of the X-ray radiation, K is the shape coefficient equal to 0.9 and is the diffraction angle. The morphology of the deposited sintered HAP/Lig coatings on titanium were studied by scanning electron microscopy (SEM) using a JEOL JSM-5800 instrument.
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The thermal behavior of the non-sintered HAP/Lig coatings, dried at room temperature and then scraped from titanium substrate, was examined by non-isothermal thermogravimetric analysis (TGA) using a Mettler Toledo instrument (TGA/SDTA851e). The thermogravimetric (TG) and differential thermogravimetric (DTG) curves were recorded from 25 to 1000 ◦ C at a heating rate of 20 ◦ C/min, under a N2 atmosphere (flow rate 50 ml/min). For each experiment, about 5 mg of oven-dried samples were used. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) measurements were used to identify and verify the presence of specific functional groups on the surface of non-sintered and sintered HAP/Lig coatings. ATR-FT-IR was realized using a Perkin Elmer Spectrum 400 FT-IR spectrometer in the wavenumber range from 525 to 4000 cm−1 . The measurements were performed at a resolution of 4 cm−1 and 64 scans were accumulated. In order to identify the elements present at the surface of the HAP/Lig coatings, X-ray photoelectron spectroscopy (XPS, Kratos Axis-Ultra) was employed over a wide range of 0–1100 eV under a high vacuum of 5 × 10−10 Torr. The take-off angle (h) of the emitted photoelectrons was adjusted to 30◦ with respect to the surface normal. The XPS spectra were recorded using a monochromatic Al–K˛ source operating at 300 W and pass energy of 160 eV for the low-resolution survey scans. The atomic compositions of the coatings surfaces were determined from high-resolution scans for C 1s, Ca 2p, P 2p and O 1s at a pass energy of 20 eV with a 0.5 eV resolution, while pass energy of 40 eV with a 0.6 eV resolution was used for Ti 2p. The HAP/Lig coatings with the minimum and maximum lignin concentration (1 and 10 wt% Lig) were chosen for the cytotoxicity test. Cell survival was determined using the method based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to assess the activity of living cells by their mitochondrial dehydrogenase activity [30,31]. The nutrient medium used in the experiments was RPM1 1640 medium supplemented with 10% heat-inactivated bovine serum, penicillin (100 IU/ml), streptomycin (100 g/ml), l-glutamine (3 mM) and 25 mM Hepes. Peripheral blood mononuclear cells (PBMC) stimulated to proliferation with mitogen phytohemagglutinin (PHA) were used as the experimental in vitro cytotoxicity model. Fig. 1. XRD patterns of: (a) non-sintered and (b) sintered HAP/Lig coatings with 0.5 wt% Lig.
3. Results and discussion 3.1. XRD analysis XRD analysis was performed to determine the phase composition and structure of the electrophoretically deposited HAP/Lig coatings with different lignin concentrations before and after sintering. The XRD patterns of the used HAP powder, a pure HAP coating and a HAP/Lig coating with 1 wt% Lig were previously reported [21]. The XRD patterns of non-sintered and sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig are represented in Figs. 1, 2 and 3, respectively. All the peaks in XRD patterns of the non-sintered HAP/Lig coatings (Figs. 1a, 2a and 3a) correspond to the JCPDS pattern No. 09–0432 for hydroxyapatite, confirming the identity of HAP in the studied coatings. The additional peaks in the diffractograms originate from the Ti substrate and they are indicated in Figs. 1a, 2a and 3a. After sintering, the diffraction peaks of HAP/Lig coatings become sharper and of higher intensity with a decrease in peak width (Figs. 1b, 2b and 3b), which all indicate that the sintered coatings had a better crystallinity. The sintering depends on the characteristics of the initial HAP powder, and the smaller particles have a tendency to aggregate in order to minimize their high free surface energy, resulting in
densification and an increase in the grain size [32,33]. Due to the nano-size of the HAP particles, a sintering temperature of 900 ◦ C was successfully applied for the thermal treatment of the composite HAP/Lig coatings, although the usually applied sintering temperature is in the range between 1000 and 1300 ◦ C [34]. The appearance of new peaks in the XRD pattern of the sintered HAP/Lig coating with 0.5 wt% Lig (Fig. 1b) indicates that partial decomposition of the HAP occurred during sintering. While the main crystalline phase of sintered coating was still HAP, the observed new diffraction peaks indicated the formation of crystalline phases of CaO, CaCO3 and TiP (Fig. 1b). It can be proposed that reaction of CaO, generated during the sintering of HAP/Lig coating, with traces of atmospheric water and CO2 yields Ca(OH)2 and CaCO3 , respectively [35]. The diffusion of phosphorous ions into the Ti surface, as a result of HAP decomposition, was evidenced by the presence of the not very strong but specific peak of TiP (Fig. 1b). It was reported that during the thermal process, the diffusion of calcium (limited) and phosphorous (profuse diffusion) ions into the Ti substrate can occur, resulting in the decomposition of the HAP block [36]. But, on the other hand the specific diffraction peak for ˇ-Ca3 (PO4 )2 , (TCP – tricalcium phosphate) at 2 = 30.7◦ [37] was absent. The partial HAP decomposition was also reported for a sintered pure HAP coating [21].
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Fig. 2. XRD patterns of: (a) non-sintered and (b) sintered HAP/Lig coatings with 3 wt% Lig.
The XRD patterns of the sintered HAP/Lig coatings containing 3 and 10 wt% Lig did not show any new crystalline phase (Figs. 2b and 3b, respectively), indicating that the phase composition remained the same as before sintering, as was previously observed for a HAP/Lig coating containing 1 wt% Lig [21]. Comparing the XRD patterns of all sintered HAP/Lig coatings, it could be concluded that the decomposition of HAP lattice did not occur when the lignin concentration was higher than 0.5 wt%; this means that higher lignin concentrations protect the HAP lattice during sintering. The mean crystallite domain size (Dp ) of the HAP/Lig coatings with different lignin concentrations was calculated using the Scherer equation (1) from the diffraction peak that corresponds to (0 0 2) reflection at about 2 ≈ 26◦ The values of mean crystallite domain size were calculated to be 35, 39 and 36 nm for coatings with 0.5, 3 and 10 wt% lignin concentration, respectively, indicating that mean crystallite domain size does not depend on the lignin concentration. 3.2. SEM analysis The surface morphologies of the electrodeposited HAP/Lig coatings containing different concentrations of lignin after thermal treatment at 900 ◦ C were analyzed by SEM. The fractured surfaces
Fig. 3. XRD patterns of: (a) non-sintered and (b) sintered HAP/Lig coatings with 10 wt% Lig.
of sintered HAP/Lig composite coatings containing 0.5, 3 and 10 wt% Lig can be seen in Fig. 4a, b and c, respectively. The cracks on coatings surfaces may be due to mechanical (HAP–lignin interactions) and thermal stresses (sintering). Since the SEM micrograph of the sintered HAP/Lig coating containing 1 wt% Lig revealed a homogenous surface without fractures [21]. These results could indicate that the optimal concentration to obtain coatings with a smooth surface is 1 wt% Lig. 3.3. TG/DTG analysis The TG curves of the non-sintered HAP/Lig coatings containing 0.5, 3 and 10 wt% Lig, obtained in the temperature range between 25 and 1000 ◦ C, are shown in Fig. 5a. Three stages can be distinguished in Fig. 5a. The first stage was observed from 25 to 200 ◦ C with sharp peaks on the DTG curves at 53, 55 and 63 ◦ C for coatings with 0.5, 3 and 10 wt% Lig, respectively (Fig. 5b). This stage corresponds to desorption of water molecules adsorbed on the surface of the crystallites of the HAP/Lig coatings [10]. The second stage of weight loss of the TG curves was observed between 200 and 600 ◦ C (Fig. 5a), with sharp peaks on the DTG curves at 227, 239 and 256 ◦ C (Fig. 5b), which correspond to the thermal decomposition of lignin [21,38]. The peaks at DTG curves
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Fig. 4. SEM micrographs of sintered HAP/Lig coatings with: (a) 0.5, (b) 3 and (c) 10 wt% Lig.
at around 353, 383 and 392 ◦ C (Fig. 5b) can be attributed to dehydroxylation of HAP. The third stage of weight loss of the TG curves was observed between 600 and 1000 ◦ C (Fig. 5a) without distinguishable peaks on the DTG curves (Fig. 5b). This phase could still be attributed to dehydroxylation or to the early slow decomposition of HAP and of the remaining lignin. The total weight loss in the temperature range of 25–1000 ◦ C was 6, 7.5 and 11.8 wt% for the coatings containing 0.5, 3 and 10 wt% Lig, respectively, suggesting that increasing lignin concentration decreased the thermal stability of the HAP/Lig coatings. In other words, increasing HAP concentration increased the thermal stability of the HAP/Lig coatings.
3.4. ATR-FT-IR analysis The ATR-FTIR spectra of the non-sintered and sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig are presented in Figs. 6 and 7, respectively. In the region of 3000–3700 cm−1 , the broad band in all spectra (not shown) is attributed to water absorption [37]. The spectra of non-sintered HAP/Lig coatings shown in Fig. 6, exhibit bands typical for the PO4 3− group. The band at 960 cm−1 is assigned to 1 PO4 3− , and the bands at 1030 and 1090 cm−1 belong to 3 PO4 3− (Fig. 6b). In addition, the two bands at 560 and 600 cm−1 (Fig. 6d) are ascribed to the PO4 3− group (4 PO4 3− ) [39], and the characteristic band at 630 cm−1 corresponds to the vibration of structural OH− groups [4]. The slight shoulder between 870 and 885 cm−1 (Fig. 6c) can be attributed to the C H deformation vibration of C H bonds in the aromatic rings, which confirm the presence of lignin
[40]. This implies that lignin in HAP/Lig coating does not change the formation and structure of HAP. In Fig. 6a, the peaks detected at 1420 and 1450 cm−1 are attributed to the methoxy group of lignin [41]. According to the literature, bands characteristic for C H stretching vibrations are present at 2939, 2881, 1460 and 1425 cm−1 [41]. Most of lignin hydroxyl groups are phenolic hydroxyl groups, which have a strong ability to form hydrogen bonds with the carbonyl groups; this formation of hydrogen bonds would induce an obvious shift of the band to lower wavenumbers. This could be taken as evidence for the formation of intermolecular hydrogen bonds. The band at 630 cm−1 (Fig. 6d), ascribed to (O H) vibrations in OH− groups from HAP, occurs at a lower wavenumber than the expected (650 cm−1 ), confirming that these groups are involved in intermolecular hydrogen bonds between HAP and Lig [42]. According to the literature, an absorption peak at 1101 cm−1 is due to (P O) stretching vibrations of the phosphorous group [43]. Therefore, it could be proposed that inter hydrogen bonds (P O · · · OH) between OH− groups from lignin and PO4 3− groups from HAP were established as evidenced by this band appearing at a new wavenumber, 1090 cm−1 (Fig. 6b). This observation proved our previously proposed model [21]. Degradation of lignin during sintering involves fragmentation of inter-unit linkages between phenolic hydroxyl and benzylic hydroxyl groups, releasing monomeric cinnamic alcohols into the vapor phase, with the remaining non-volatilized fraction having a highly condensed aromatic structure [44]. After sintering, there were visible differences in the shapes of the spectra in the ranges 1350–1550 and 850–900 cm−1 between the non-sintered HAP/Lig (Fig. 6a and c) and the sintered HAP/Lig coatings (Fig. 7a and c). Thus, in the case of sintered HAP/Lig coatings (Fig. 7a and c),
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Table 1 Quantitative analysis data of the XPS spectra. HAP/Lig, wt% Lig 0.5 3 10
Non-sintered Sintered Non-sintered Sintered Non-sintered Sintered
Ca
P
O
C
Ti
Na
Ba
F
Cl
S
19.1 18.4 18.4 18.8 15.8 17.1
11.3 7.9 12.0 10.8 10.3 9.6
58.3 56.4 55.5 55.2 51.1 52.2
8.2 15.9 11.7 12.9 21.3 18.9
– – – – – –
0.3 – 0.2 – 0.14 –
0.1 0.2 0.1 0.1 0.1 0.3
0.2 0.2 – 0.3 – 0.1
0.5 0.2 1.1 0.6 0.5 0.6
1.6 0.9 1.0 1.2 0.7 1.2
the band intensities at 1410 and 1460 cm−1 decreased while no band shifts could be observed. Weak carbonate bands in the range 865–880 cm−1 were observed only for the sintered HAP/Lig coating with 0.5 wt% Lig (Fig. 7c), which is indicative of HAP decomposition during sintering. Comparing the ATR-FTIR results obtained here for 0.5, 3 and 10 wt% Lig, as well as for 1 wt% Lig [21], it could be concluded that decomposition of HAP during sintering does not occur for coatings with lignin concentration higher than 0.5 wt%. In other words,
lignin concentrations higher than 0.5 wt% prevent HAP decomposition and/or diffusion of phosphorous ions into the Ti surface due to the established hydrogen bonds. These results are in accordance with the results of the XRD analysis.
3.5. XPS analysis XPS analysis was performed on deposited HAP/Lig coatings and the qualitative analysis revealed certain differences between the non-sintered and sintered coatings with 0.5, 3 and 10 wt% Lig. The quantitative analysis data of the XPS spectra obtained from high-resolution measurement are presented in Table 1. The carbon content of the non-sintered coatings increased with increasing Lig concentration, which confirmed that lignin was bonded with HAP lattice, as was seen from the TG curves (Fig. 5a). However, the highest increase in the carbon content after sintering was determined for the HAP/Lig coating with 0.5 wt% Lig, which could be explained by carbon from the CaCO3 formed by reaction between CaO with atmospheric CO2 . It could also be noticed that CaO and CaCO3 were detected after sintering in the XRD pattern of the same coatings, confirming the HAP decomposition (Fig. 1b). The decrease in carbon content after sintering for the coating with 10 wt% Lig (Table 1) indicates the highest weight loss of lignin, which was also registered by TG analysis (Fig. 5a). The Ca/P ratio, presented in Table 2, varied in the range of 1.53–1.69 for the non-sintered HAP/Lig coatings, which is similar to Ca/P ratio for stoichiometric HAP (1.67). According to the literature [45], stable HAP phases correspond to a Ca/P ratio within a range of 1.3–1.8. It could be observed that the Ca/P ratios of the sintered HAP/Lig coatings were higher than those of the non-sintered coatings. The highest increase in Ca/P ratio after sintering, recorded for the HAP/Lig coating with 0.5 wt% Lig, refers to HAP decomposition during sintering (Table 2), which was also confirmed by appearance of a TiP peak in XRD pattern (Fig. 1b). It could be concluded that lignin limited the decomposition of the HAP lattice during the sintering of the coatings with 3 and 10 wt% Lig, which is indicated by the smaller increase in carbon content (Table 1) and smaller Ca/P ratio (Table 2), compared to the HAP/Lig coating with 0.5 wt% Lig. This was also confirmed by the XRD and ATR-FT-IR results.
Table 2 Ca/P ratio for the HAP/Lig coatings with different Lig concentrations. HAP/Lig, wt% Lig 0.5 3 Fig. 5. TG curves (a) and differential TG (DTG) curves (b) of non-sintered HAP/Lig coatings with: 0.5, 3 and 10 wt% Lig.
10
Ca/P Non-sintered Sintered Non-sintered Sintered Non-sintered Sintered
1.69 2.33 1.53 1.74 1.53 1.78
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Fig. 6. ATR-FT-IR spectra of non-sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig, in the wavenumber range 525–1550 cm−1 .
3.6. Cytotoxicity Cell survival (S, %) is defined as the ratio of the number of cells grown in nutrient medium with coating and the number of cells grown in control wells containing nutrient medium without coating, multiplied by 100. As the number of live cells is directly proportional to the absorbance of live metabolically active MTTtreated cells, for the calculation of cell survival, absorbance of the newly formed formazan was used instead of the number of live cells: S(%) =
Au × 100 Ak
(2)
where Au is the absorbance of the cells grown in the presence of a coating and Ak is the absorbance of the cells of the control sample. Experiments were performed in triplicate. The results are reported as the average value ± standard deviation (SD). The survival of PBMC stimulated to proliferate with mitogen phytohemagglutinin (PHA) in control sample and in the presence of
sintered HAP and HAP/Lig coatings (1 and 10 wt% Lig) were investigated 72 h after seeding. The results presented in Table 3 show that the survival of the PHA-stimulated PBMC did not decrease significantly with increasing lignin concentration. MTT results indicate that HAP and both HAP/Lig coatings could induce a mild decrease in survival of healthy immunocompetent PHA-stimulated PBMC but that all the results were similar to that of the control sample (S = 100%). According to literature [46], HAP coating and HAP/Lig coating with 1 wt% Lig can be classified as non-toxic, while HAP/Lig coating with 10 wt% Lig as slightly cytotoxic. The reason for the slight lignin cytotoxicity could be due to its known absorption capability, i.e. it could slightly non-specifically absorb some of micronutrient constituents needed for sustaining tested PBMC proliferation, but also with lignin antioxidant activity as it was shown earlier doing the tests on human keratinocytes and on mouse fibroblasts [47]. On the other hand, one can speculate that lignin structures became more condensed upon sintering, which could have led to formation of highly condensed
Table 3 Cell survival of PBMC cells stimulated to proliferation in the presence of sintered HAP and HAP/Lig coatings. Cell type
PHA-stimulated peripheral blood mononuclear cells (PBMC)
Material
HAP coating
HAP/Lig coating, 1 wt% Lig
HAP/Lig coating, 10 wt% Lig
Cell survival (S), % Classification
93.4 ± 4.0 Non-cytotoxic
90.4 ± 8.2 Non-cytotoxic
83.7 ± 5.8 Slightly cytotoxic
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Fig. 7. ATR-FT-IR spectra of sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig, in the wavenumber range 525–1550 cm−1 .
aromatic structures, the type of structures known for their toxicity.
4. Conclusions Composite HAP/Lig coatings were successfully produced by EPD and sintered at a low sintering temperature of 900 ◦ C. The XRD and ATR-FTIR analyses showed that partial decomposition of the HAP lattice occurred only during the sintering of the HAP/Lig coating with 0.5 wt% Lig, while lignin concentrations higher than 0.5 wt% protected the HAP lattice. The TGA results indicated that with increasing lignin concentration, the thermal stability of the coatings decreased and also show that the stability of the HAP increased. The XPS results showed that the Ca/P ratio for all non-sintered HAP/Lig coatings was similar to the Ca/P ratio for stoichiometric HAP (1.67), independent of the lignin concentration. Moreover, ATR-FTIR proved that hydrogen bonds were formed between lignin and the HAP lattice. The results of the cytotoxicity determination by the MTT test indicated that HAP/Lig coating with 1 wt% Lig could be a promising non-toxic biomaterial for bone tissue engineering.
Acknowledgements This research was financed by the Ministry of Education and Science of the Republic of Serbia, contracts Nos. III 45019, OI 175011, III 45015 and by the National Sciences and Engineering Research Council of Canada (NSERC). The authors would like to thank Yves
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Contents lists available at SciVerse ScienceDirect
Progress in Organic Coatings journal homepage: www.elsevier.com/locate/porgcoat
Review
The effect of lignin on the structure and characteristics of composite coatings electrodeposited on titanium Sanja Erakovic´ a , Djordje Veljovic´ a , Papa N. Diouf b , Tatjana Stevanovic´ c , Miodrag Mitric´ d , ´ ´ Djordje Janackovi c´ a , Ivana Z. Matic´ e , Zorica D. Juranic´ e , Vesna Miˇskovic-Stankovi c´ a,∗ a
Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia Service de recherche et d’expertise en transformation des produits forestiers, 25 rue Armand-Sinclair, porte 5, Amqui, Canada c Département des sciences du bois et de la forêt, Université Laval, 2425 rue de la Terrasse, Québec, Canada d Vinˇca Institute of Nuclear Sciences, University of Belgrade, Mike Petrovi´ca Alasa 12–14, 11000 Belgrade, Serbia e Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 3 July 2012 Accepted 13 July 2012 Available online 4 August 2012 Keywords: Electrophoretic deposition Composite coating Organosolv lignin Hydroxyapatite Titanium Cytotoxicity
a b s t r a c t Composite coatings based on lignin, obtained by electrophoretic deposition (EPD) on titanium, were investigated. The aim of this work was to produce hydroxyapatite/lignin (HAP/Lig) coatings on titanium and to investigate the effect of the lignin concentration on microstructure, morphology, phase composition, thermal behavior and cytotoxicity of the HAP/Lig coatings. An organosolv lignin was used for the production of the composite coatings studied in this research. The properties of HAP/Lig coatings were characterized using X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) and X-ray photoelectron spectroscopy (XPS), as well as the MTT test of cytotoxicity. The results showed that higher lignin concentrations protected the HAP lattice during sintering, thereby improving the stability of the HAP/Lig coatings. The cell survival of peripheral blood mononuclear cells (PBMC) after proliferation indicates that the HAP/Lig coating with 1 wt% Lig electrodeposited on titanium was non-toxic with significant promise as a potential bone-repair material. © 2012 Elsevier B.V. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Alcell lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Nano-sized HAP powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Electrophoretic deposition of composite HAP/Lig coatings on titanium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Characterization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. XRD analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. SEM analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. TG/DTG analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. ATR-FT-IR analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. XPS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +381 11 3303 737; fax: +381 11 3370 387. ´ ´ E-mail address: [email protected] (V. Miˇskovic-Stankovi c). 0300-9440/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.porgcoat.2012.07.005
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1. Introduction Electrophoretic deposition (EPD) is gaining increasing attention as a powerful method for the formation of both uniform thin and thick films on substrates of complex geometry [1–12]. This method is worldwide used for the deposition of organic protective coatings [6–9], ceramic coatings [10–12], bioceramic coatings [2–4,13] and composite coatings [5,14]. In the past few decades, EPD has become an attractive technique for the formation of bioactive ceramic coatings, such as hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HAP), on metallic implants [2]. Titanium and some of its alloys are most often the material of choice for metallic implants due to their attributes of strength, stiffness, toughness, impact resistance and corrosion resistance [15–17]. Titanium has proven itself to be potentially a very suitable material for orthopedic and dental implants, due to its excellent corrosion resistance and biocompatibility. Calcium phosphate coated on metal implants provides the necessary porosity for bone in-growths, therefore HAP is often used as a coating material [17]. Hence, a good combination of the biocompatibility of hydroxyapatite and the excellent mechanical properties of metals is considered a promising approach to fabricate more suitable bone implants. However, the application of pure electrodeposited HAP coatings on metal implants has been limited due to problems such as low adhesion. In order to improve the mechanical properties, adhesion and the brittleness of HAP coatings, composite HAP coatings containing biopolymers have become of great interest [18–20]. As complex natural polymer networks composed primarily of phenolic moieties, lignins have a wide variety of chemical bonds [21,22]. Among the functional groups present in lignin, the most reactive chemical sites are phenolic hydroxyl groups [23]. Other major chemical functional groups in lignins include methoxyl, carbonyl and carboxyl groups, depending on the plant origin and the applied pulping processes [24]. Organosolv lignins are being examined because they show significantly better solubility and thermal properties than sulfite or kraft lignins [25]. The organosolv processes are convenient because of the greater simplicity of the chemical recovery system, since only the solvent has to be recovered by rectification of the black liquors [23]. In contrast to Kraft and sulfite lignin, Alcell (organosolv) lignin derives from a process using ethanol as the only pulping chemical and consists of low molecular weight phenol fragments with enhanced hydrophobicity [22]. Hence, this organosolv lignin, in its purified form, possesses a chemical structure different to that of native lignin. The incorporation of lignin is interesting in medical applications because of its thermal stability and biocompatibility in different materials [26–28]. In this work, the influence of the lignin concentration on the microstructure, morphology, phase composition, thermal behavior and cytotoxicity of composite HAP/Lig coatings electrodeposited on titanium was investigated.
2. Experimental 2.1. Materials 2.1.1. Alcell lignin The lignin powder used without further purification was Alcell lignin from Repap Enterprises Inc. (Stamford, CT) that was extracted from a mixture of hardwoods (maple, birch and poplar) by an organosolv process using aqueous ethanol. The characteristics of the employed AlcellTM lignin were reported in a previous paper [21].
2.1.2. Nano-sized HAP powder A nano-sized HAP powder was obtained using a modified chemical precipitation method, by the reaction of calcium oxide (obtained by calcination of CaCO3 for 5 h at 1000 ◦ C in air) with phosphoric acid, as described in a previous paper [21]. Small portions of the resulting calcium oxide were mixed and stirred with distilled water for 10 min. A calculated amount of phosphoric acid was added dropwise to the calcium hydroxide suspension until the pH reached the value 7.4–7.6. The obtained suspension was heated to 94 ± 1 ◦ C for 30 min and stirred for a further 30 min. After sedimentation, the upper clear solution layer was decanted. The suspension was then spray dried at 120 ± 5 ◦ C into a granulated powder. It was determined by TEM analysis that the mean particle size of the rod-shaped HAP powder was between 50 and 100 nm [29]. 2.2. Electrophoretic deposition of composite HAP/Lig coatings on titanium Electrophoretic deposition was performed from ethanol suspensions containing 1.0 g of nano-sized HAP powder and 0.5–10 wt% of lignin powder in 100 ml of absolute ethanol after ultrasonic treatment for 15 min to obtain homogeneous and stable suspensions. To increase the stability of the suspensions, HCl was added until a pH value of 2.00 was attained. Prior to electrodeposition, the mixtures were ultrasonicated for 30 min to obtain a homogeneous suspension of the particles. A three-electrode cell arrangement was used for the cathodic electrodeposition. The working electrode used as a substrate for the deposition of HAP and HAP/Lig coatings was a titanium plate (65 mm × 5 mm × 0.25 mm, Aldrich, purity 99.7%). The Ti surface was pretreated by polishing with grit emery paper, followed by wet polishing with 0.3 m alumina. After polishing, the titanium plates were degreased with acetone and then with ethanol for 15 min in an ultrasonic bath. The counter electrodes were two platinum panels, placed parallel to the Ti electrode at a distance of 1.5 cm. The HAP and HAP/Lig coatings were obtained on titanium from ethanol HAP and HAP/Lig suspensions using the constant voltage method at a deposition voltage of 60 V for a deposition time of 45 s, at room temperature. The deposited HAP and HAP/Lig coatings were air dried at room temperature and then sintered at 900 ◦ C for 30 min, with the initial heating rate of 16 ◦ C/min, under an argon atmosphere. Before sintering, deoxidization the coatings were deoxidized at 200 ◦ C under an argon atmosphere for 45 min. 2.3. Characterization techniques The phase composition of HAP/Lig coatings was investigated by X-ray diffraction (XRD) analysis using a Philips PW 1051 Powder ˚ The Diffractometer with Ni filtered CuK˛ radiation ( = 1.5418 A). diffraction intensity was measured using the scan-step technique in the 2 range 8–80◦ with a scanning step width of 0.05◦ and exposition time of 50 s per step. The phase analysis was realized using the PDF-2 data base with a commercially available computer program, EVA V.9.0. The average crystallite domain size (Dp ) was calculated from the half height width (ˇ1/2 ) of the XRD reflection of the (0 0 2) plane (at 2 = 25.8◦ ), using the Scherer equation (1): Dp =
K ˇ1/2 cos
(1)
where is the wavelength of the X-ray radiation, K is the shape coefficient equal to 0.9 and is the diffraction angle. The morphology of the deposited sintered HAP/Lig coatings on titanium were studied by scanning electron microscopy (SEM) using a JEOL JSM-5800 instrument.
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The thermal behavior of the non-sintered HAP/Lig coatings, dried at room temperature and then scraped from titanium substrate, was examined by non-isothermal thermogravimetric analysis (TGA) using a Mettler Toledo instrument (TGA/SDTA851e). The thermogravimetric (TG) and differential thermogravimetric (DTG) curves were recorded from 25 to 1000 ◦ C at a heating rate of 20 ◦ C/min, under a N2 atmosphere (flow rate 50 ml/min). For each experiment, about 5 mg of oven-dried samples were used. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) measurements were used to identify and verify the presence of specific functional groups on the surface of non-sintered and sintered HAP/Lig coatings. ATR-FT-IR was realized using a Perkin Elmer Spectrum 400 FT-IR spectrometer in the wavenumber range from 525 to 4000 cm−1 . The measurements were performed at a resolution of 4 cm−1 and 64 scans were accumulated. In order to identify the elements present at the surface of the HAP/Lig coatings, X-ray photoelectron spectroscopy (XPS, Kratos Axis-Ultra) was employed over a wide range of 0–1100 eV under a high vacuum of 5 × 10−10 Torr. The take-off angle (h) of the emitted photoelectrons was adjusted to 30◦ with respect to the surface normal. The XPS spectra were recorded using a monochromatic Al–K˛ source operating at 300 W and pass energy of 160 eV for the low-resolution survey scans. The atomic compositions of the coatings surfaces were determined from high-resolution scans for C 1s, Ca 2p, P 2p and O 1s at a pass energy of 20 eV with a 0.5 eV resolution, while pass energy of 40 eV with a 0.6 eV resolution was used for Ti 2p. The HAP/Lig coatings with the minimum and maximum lignin concentration (1 and 10 wt% Lig) were chosen for the cytotoxicity test. Cell survival was determined using the method based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to assess the activity of living cells by their mitochondrial dehydrogenase activity [30,31]. The nutrient medium used in the experiments was RPM1 1640 medium supplemented with 10% heat-inactivated bovine serum, penicillin (100 IU/ml), streptomycin (100 g/ml), l-glutamine (3 mM) and 25 mM Hepes. Peripheral blood mononuclear cells (PBMC) stimulated to proliferation with mitogen phytohemagglutinin (PHA) were used as the experimental in vitro cytotoxicity model. Fig. 1. XRD patterns of: (a) non-sintered and (b) sintered HAP/Lig coatings with 0.5 wt% Lig.
3. Results and discussion 3.1. XRD analysis XRD analysis was performed to determine the phase composition and structure of the electrophoretically deposited HAP/Lig coatings with different lignin concentrations before and after sintering. The XRD patterns of the used HAP powder, a pure HAP coating and a HAP/Lig coating with 1 wt% Lig were previously reported [21]. The XRD patterns of non-sintered and sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig are represented in Figs. 1, 2 and 3, respectively. All the peaks in XRD patterns of the non-sintered HAP/Lig coatings (Figs. 1a, 2a and 3a) correspond to the JCPDS pattern No. 09–0432 for hydroxyapatite, confirming the identity of HAP in the studied coatings. The additional peaks in the diffractograms originate from the Ti substrate and they are indicated in Figs. 1a, 2a and 3a. After sintering, the diffraction peaks of HAP/Lig coatings become sharper and of higher intensity with a decrease in peak width (Figs. 1b, 2b and 3b), which all indicate that the sintered coatings had a better crystallinity. The sintering depends on the characteristics of the initial HAP powder, and the smaller particles have a tendency to aggregate in order to minimize their high free surface energy, resulting in
densification and an increase in the grain size [32,33]. Due to the nano-size of the HAP particles, a sintering temperature of 900 ◦ C was successfully applied for the thermal treatment of the composite HAP/Lig coatings, although the usually applied sintering temperature is in the range between 1000 and 1300 ◦ C [34]. The appearance of new peaks in the XRD pattern of the sintered HAP/Lig coating with 0.5 wt% Lig (Fig. 1b) indicates that partial decomposition of the HAP occurred during sintering. While the main crystalline phase of sintered coating was still HAP, the observed new diffraction peaks indicated the formation of crystalline phases of CaO, CaCO3 and TiP (Fig. 1b). It can be proposed that reaction of CaO, generated during the sintering of HAP/Lig coating, with traces of atmospheric water and CO2 yields Ca(OH)2 and CaCO3 , respectively [35]. The diffusion of phosphorous ions into the Ti surface, as a result of HAP decomposition, was evidenced by the presence of the not very strong but specific peak of TiP (Fig. 1b). It was reported that during the thermal process, the diffusion of calcium (limited) and phosphorous (profuse diffusion) ions into the Ti substrate can occur, resulting in the decomposition of the HAP block [36]. But, on the other hand the specific diffraction peak for ˇ-Ca3 (PO4 )2 , (TCP – tricalcium phosphate) at 2 = 30.7◦ [37] was absent. The partial HAP decomposition was also reported for a sintered pure HAP coating [21].
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Fig. 2. XRD patterns of: (a) non-sintered and (b) sintered HAP/Lig coatings with 3 wt% Lig.
The XRD patterns of the sintered HAP/Lig coatings containing 3 and 10 wt% Lig did not show any new crystalline phase (Figs. 2b and 3b, respectively), indicating that the phase composition remained the same as before sintering, as was previously observed for a HAP/Lig coating containing 1 wt% Lig [21]. Comparing the XRD patterns of all sintered HAP/Lig coatings, it could be concluded that the decomposition of HAP lattice did not occur when the lignin concentration was higher than 0.5 wt%; this means that higher lignin concentrations protect the HAP lattice during sintering. The mean crystallite domain size (Dp ) of the HAP/Lig coatings with different lignin concentrations was calculated using the Scherer equation (1) from the diffraction peak that corresponds to (0 0 2) reflection at about 2 ≈ 26◦ The values of mean crystallite domain size were calculated to be 35, 39 and 36 nm for coatings with 0.5, 3 and 10 wt% lignin concentration, respectively, indicating that mean crystallite domain size does not depend on the lignin concentration. 3.2. SEM analysis The surface morphologies of the electrodeposited HAP/Lig coatings containing different concentrations of lignin after thermal treatment at 900 ◦ C were analyzed by SEM. The fractured surfaces
Fig. 3. XRD patterns of: (a) non-sintered and (b) sintered HAP/Lig coatings with 10 wt% Lig.
of sintered HAP/Lig composite coatings containing 0.5, 3 and 10 wt% Lig can be seen in Fig. 4a, b and c, respectively. The cracks on coatings surfaces may be due to mechanical (HAP–lignin interactions) and thermal stresses (sintering). Since the SEM micrograph of the sintered HAP/Lig coating containing 1 wt% Lig revealed a homogenous surface without fractures [21]. These results could indicate that the optimal concentration to obtain coatings with a smooth surface is 1 wt% Lig. 3.3. TG/DTG analysis The TG curves of the non-sintered HAP/Lig coatings containing 0.5, 3 and 10 wt% Lig, obtained in the temperature range between 25 and 1000 ◦ C, are shown in Fig. 5a. Three stages can be distinguished in Fig. 5a. The first stage was observed from 25 to 200 ◦ C with sharp peaks on the DTG curves at 53, 55 and 63 ◦ C for coatings with 0.5, 3 and 10 wt% Lig, respectively (Fig. 5b). This stage corresponds to desorption of water molecules adsorbed on the surface of the crystallites of the HAP/Lig coatings [10]. The second stage of weight loss of the TG curves was observed between 200 and 600 ◦ C (Fig. 5a), with sharp peaks on the DTG curves at 227, 239 and 256 ◦ C (Fig. 5b), which correspond to the thermal decomposition of lignin [21,38]. The peaks at DTG curves
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Fig. 4. SEM micrographs of sintered HAP/Lig coatings with: (a) 0.5, (b) 3 and (c) 10 wt% Lig.
at around 353, 383 and 392 ◦ C (Fig. 5b) can be attributed to dehydroxylation of HAP. The third stage of weight loss of the TG curves was observed between 600 and 1000 ◦ C (Fig. 5a) without distinguishable peaks on the DTG curves (Fig. 5b). This phase could still be attributed to dehydroxylation or to the early slow decomposition of HAP and of the remaining lignin. The total weight loss in the temperature range of 25–1000 ◦ C was 6, 7.5 and 11.8 wt% for the coatings containing 0.5, 3 and 10 wt% Lig, respectively, suggesting that increasing lignin concentration decreased the thermal stability of the HAP/Lig coatings. In other words, increasing HAP concentration increased the thermal stability of the HAP/Lig coatings.
3.4. ATR-FT-IR analysis The ATR-FTIR spectra of the non-sintered and sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig are presented in Figs. 6 and 7, respectively. In the region of 3000–3700 cm−1 , the broad band in all spectra (not shown) is attributed to water absorption [37]. The spectra of non-sintered HAP/Lig coatings shown in Fig. 6, exhibit bands typical for the PO4 3− group. The band at 960 cm−1 is assigned to 1 PO4 3− , and the bands at 1030 and 1090 cm−1 belong to 3 PO4 3− (Fig. 6b). In addition, the two bands at 560 and 600 cm−1 (Fig. 6d) are ascribed to the PO4 3− group (4 PO4 3− ) [39], and the characteristic band at 630 cm−1 corresponds to the vibration of structural OH− groups [4]. The slight shoulder between 870 and 885 cm−1 (Fig. 6c) can be attributed to the C H deformation vibration of C H bonds in the aromatic rings, which confirm the presence of lignin
[40]. This implies that lignin in HAP/Lig coating does not change the formation and structure of HAP. In Fig. 6a, the peaks detected at 1420 and 1450 cm−1 are attributed to the methoxy group of lignin [41]. According to the literature, bands characteristic for C H stretching vibrations are present at 2939, 2881, 1460 and 1425 cm−1 [41]. Most of lignin hydroxyl groups are phenolic hydroxyl groups, which have a strong ability to form hydrogen bonds with the carbonyl groups; this formation of hydrogen bonds would induce an obvious shift of the band to lower wavenumbers. This could be taken as evidence for the formation of intermolecular hydrogen bonds. The band at 630 cm−1 (Fig. 6d), ascribed to (O H) vibrations in OH− groups from HAP, occurs at a lower wavenumber than the expected (650 cm−1 ), confirming that these groups are involved in intermolecular hydrogen bonds between HAP and Lig [42]. According to the literature, an absorption peak at 1101 cm−1 is due to (P O) stretching vibrations of the phosphorous group [43]. Therefore, it could be proposed that inter hydrogen bonds (P O · · · OH) between OH− groups from lignin and PO4 3− groups from HAP were established as evidenced by this band appearing at a new wavenumber, 1090 cm−1 (Fig. 6b). This observation proved our previously proposed model [21]. Degradation of lignin during sintering involves fragmentation of inter-unit linkages between phenolic hydroxyl and benzylic hydroxyl groups, releasing monomeric cinnamic alcohols into the vapor phase, with the remaining non-volatilized fraction having a highly condensed aromatic structure [44]. After sintering, there were visible differences in the shapes of the spectra in the ranges 1350–1550 and 850–900 cm−1 between the non-sintered HAP/Lig (Fig. 6a and c) and the sintered HAP/Lig coatings (Fig. 7a and c). Thus, in the case of sintered HAP/Lig coatings (Fig. 7a and c),
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Table 1 Quantitative analysis data of the XPS spectra. HAP/Lig, wt% Lig 0.5 3 10
Non-sintered Sintered Non-sintered Sintered Non-sintered Sintered
Ca
P
O
C
Ti
Na
Ba
F
Cl
S
19.1 18.4 18.4 18.8 15.8 17.1
11.3 7.9 12.0 10.8 10.3 9.6
58.3 56.4 55.5 55.2 51.1 52.2
8.2 15.9 11.7 12.9 21.3 18.9
– – – – – –
0.3 – 0.2 – 0.14 –
0.1 0.2 0.1 0.1 0.1 0.3
0.2 0.2 – 0.3 – 0.1
0.5 0.2 1.1 0.6 0.5 0.6
1.6 0.9 1.0 1.2 0.7 1.2
the band intensities at 1410 and 1460 cm−1 decreased while no band shifts could be observed. Weak carbonate bands in the range 865–880 cm−1 were observed only for the sintered HAP/Lig coating with 0.5 wt% Lig (Fig. 7c), which is indicative of HAP decomposition during sintering. Comparing the ATR-FTIR results obtained here for 0.5, 3 and 10 wt% Lig, as well as for 1 wt% Lig [21], it could be concluded that decomposition of HAP during sintering does not occur for coatings with lignin concentration higher than 0.5 wt%. In other words,
lignin concentrations higher than 0.5 wt% prevent HAP decomposition and/or diffusion of phosphorous ions into the Ti surface due to the established hydrogen bonds. These results are in accordance with the results of the XRD analysis.
3.5. XPS analysis XPS analysis was performed on deposited HAP/Lig coatings and the qualitative analysis revealed certain differences between the non-sintered and sintered coatings with 0.5, 3 and 10 wt% Lig. The quantitative analysis data of the XPS spectra obtained from high-resolution measurement are presented in Table 1. The carbon content of the non-sintered coatings increased with increasing Lig concentration, which confirmed that lignin was bonded with HAP lattice, as was seen from the TG curves (Fig. 5a). However, the highest increase in the carbon content after sintering was determined for the HAP/Lig coating with 0.5 wt% Lig, which could be explained by carbon from the CaCO3 formed by reaction between CaO with atmospheric CO2 . It could also be noticed that CaO and CaCO3 were detected after sintering in the XRD pattern of the same coatings, confirming the HAP decomposition (Fig. 1b). The decrease in carbon content after sintering for the coating with 10 wt% Lig (Table 1) indicates the highest weight loss of lignin, which was also registered by TG analysis (Fig. 5a). The Ca/P ratio, presented in Table 2, varied in the range of 1.53–1.69 for the non-sintered HAP/Lig coatings, which is similar to Ca/P ratio for stoichiometric HAP (1.67). According to the literature [45], stable HAP phases correspond to a Ca/P ratio within a range of 1.3–1.8. It could be observed that the Ca/P ratios of the sintered HAP/Lig coatings were higher than those of the non-sintered coatings. The highest increase in Ca/P ratio after sintering, recorded for the HAP/Lig coating with 0.5 wt% Lig, refers to HAP decomposition during sintering (Table 2), which was also confirmed by appearance of a TiP peak in XRD pattern (Fig. 1b). It could be concluded that lignin limited the decomposition of the HAP lattice during the sintering of the coatings with 3 and 10 wt% Lig, which is indicated by the smaller increase in carbon content (Table 1) and smaller Ca/P ratio (Table 2), compared to the HAP/Lig coating with 0.5 wt% Lig. This was also confirmed by the XRD and ATR-FT-IR results.
Table 2 Ca/P ratio for the HAP/Lig coatings with different Lig concentrations. HAP/Lig, wt% Lig 0.5 3 Fig. 5. TG curves (a) and differential TG (DTG) curves (b) of non-sintered HAP/Lig coatings with: 0.5, 3 and 10 wt% Lig.
10
Ca/P Non-sintered Sintered Non-sintered Sintered Non-sintered Sintered
1.69 2.33 1.53 1.74 1.53 1.78
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Fig. 6. ATR-FT-IR spectra of non-sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig, in the wavenumber range 525–1550 cm−1 .
3.6. Cytotoxicity Cell survival (S, %) is defined as the ratio of the number of cells grown in nutrient medium with coating and the number of cells grown in control wells containing nutrient medium without coating, multiplied by 100. As the number of live cells is directly proportional to the absorbance of live metabolically active MTTtreated cells, for the calculation of cell survival, absorbance of the newly formed formazan was used instead of the number of live cells: S(%) =
Au × 100 Ak
(2)
where Au is the absorbance of the cells grown in the presence of a coating and Ak is the absorbance of the cells of the control sample. Experiments were performed in triplicate. The results are reported as the average value ± standard deviation (SD). The survival of PBMC stimulated to proliferate with mitogen phytohemagglutinin (PHA) in control sample and in the presence of
sintered HAP and HAP/Lig coatings (1 and 10 wt% Lig) were investigated 72 h after seeding. The results presented in Table 3 show that the survival of the PHA-stimulated PBMC did not decrease significantly with increasing lignin concentration. MTT results indicate that HAP and both HAP/Lig coatings could induce a mild decrease in survival of healthy immunocompetent PHA-stimulated PBMC but that all the results were similar to that of the control sample (S = 100%). According to literature [46], HAP coating and HAP/Lig coating with 1 wt% Lig can be classified as non-toxic, while HAP/Lig coating with 10 wt% Lig as slightly cytotoxic. The reason for the slight lignin cytotoxicity could be due to its known absorption capability, i.e. it could slightly non-specifically absorb some of micronutrient constituents needed for sustaining tested PBMC proliferation, but also with lignin antioxidant activity as it was shown earlier doing the tests on human keratinocytes and on mouse fibroblasts [47]. On the other hand, one can speculate that lignin structures became more condensed upon sintering, which could have led to formation of highly condensed
Table 3 Cell survival of PBMC cells stimulated to proliferation in the presence of sintered HAP and HAP/Lig coatings. Cell type
PHA-stimulated peripheral blood mononuclear cells (PBMC)
Material
HAP coating
HAP/Lig coating, 1 wt% Lig
HAP/Lig coating, 10 wt% Lig
Cell survival (S), % Classification
93.4 ± 4.0 Non-cytotoxic
90.4 ± 8.2 Non-cytotoxic
83.7 ± 5.8 Slightly cytotoxic
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Fig. 7. ATR-FT-IR spectra of sintered HAP/Lig coatings with 0.5, 3 and 10 wt% Lig, in the wavenumber range 525–1550 cm−1 .
aromatic structures, the type of structures known for their toxicity.
4. Conclusions Composite HAP/Lig coatings were successfully produced by EPD and sintered at a low sintering temperature of 900 ◦ C. The XRD and ATR-FTIR analyses showed that partial decomposition of the HAP lattice occurred only during the sintering of the HAP/Lig coating with 0.5 wt% Lig, while lignin concentrations higher than 0.5 wt% protected the HAP lattice. The TGA results indicated that with increasing lignin concentration, the thermal stability of the coatings decreased and also show that the stability of the HAP increased. The XPS results showed that the Ca/P ratio for all non-sintered HAP/Lig coatings was similar to the Ca/P ratio for stoichiometric HAP (1.67), independent of the lignin concentration. Moreover, ATR-FTIR proved that hydrogen bonds were formed between lignin and the HAP lattice. The results of the cytotoxicity determination by the MTT test indicated that HAP/Lig coating with 1 wt% Lig could be a promising non-toxic biomaterial for bone tissue engineering.
Acknowledgements This research was financed by the Ministry of Education and Science of the Republic of Serbia, contracts Nos. III 45019, OI 175011, III 45015 and by the National Sciences and Engineering Research Council of Canada (NSERC). The authors would like to thank Yves
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