Materials Science and Engineering C 36 (2014) 215–220
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Silver-doped hydroxyapatite coatings formed on Ti–6Al–4V substrates and their characterization A.A. Yanovska a,⁎, A.S. Stanislavov a, L.B. Sukhodub c, V.N. Kuznetsov a, V.Yu. Illiashenko a, S.N. Danilchenko a, L.F. Sukhodub b a b c
Institute of Applied Physics National Academy of Sciences of Ukraine, 58, Sumy 40000, Ukraine Sumy State University, Medical Institute, Ministry of Education and Science of Ukraine, R. Korsakova Str. 2, Sumy 40007, Ukraine Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine, 14-Puschinskaya St., Kharkov 61057, Ukraine
a r t i c l e
i n f o
Article history: Received 28 September 2013 Received in revised form 4 November 2013 Accepted 6 December 2013 Available online 16 December 2013 Keywords: Hydroxyapatite (HA) Chitosan (CS) Silver Antibacterial Coating
a b s t r a c t Coatings with antibacterial components for medical implants are recommended to reduce the risk of bacterial infections. Therefore hydroxyapatite (HA) coatings with addition of chitosan (CS) and silver (Ag) are proposed in this work in an attempt to resolve this problem. Ti–6Al–4V substrates were modified by a chitosan film to study the influence of surface modification on the formation of the HA–Ag and HA–CS–Ag coatings. Using a thermal substrate method, HA and HA–CS coatings doped with Ag+ were prepared at low substrate temperatures (90 °C). Coated surfaces were examined using X-ray diffraction and scanning electron microscopy. The amount of silver in the deposited coatings was analyzed by atomic absorption spectroscopy. From this study it is concluded that the substrate surface modified by a chitosan film promotes the coating formation and increases the antibacterial activity of produced coatings against a strain of Escherichia coli. The adhesion of E. coli (ATCC 25922) to sheep erythrocytes was decreased by 14% as compared with the reference samples without Ag. It could be explained by the inhibition of bacterial adhesins by Ag+ ions released. The combined action of silver ions and chitosan resulted in a 21% decrease in adhesive index. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The problem of infections related to biomedical devices and implants still remains open in spite of active research in this field [1]. Bacterial infections associated with implant materials lead to the inflammation around the implant, eventually resulting in implant loss. Bacteria may readily colonize the surfaces of synthetic medical implants [1]. The implant-associated infections may be prevented by application of antibacterial coatings based on biopolymers and inorganic compounds. Because of excellent osteoconductivity and bioactivity, hydroxyapatite (HA) is one of the most widely used biomaterials for bone reconstruction [2], as a coating material for implants to improve their biological properties [3]. Chitosan (CS) is a biopolymer produced from the deacetylation of chitin, a copolymer consisting of N-acetyl-2-amino-2-deoxy-Dglucopyranose and 2-amino-2-deoxy-D-glucopyranose, where the two types of recurring units are linked by (1 → 4)-β-glycosidic bonds [4]. Chitin and chitosan are drawing increasingly greater attention due to their biological and physicochemical properties [5]. Chitosan is the ⁎ Corresponding author at: Institute of Applied Physics National Academy of Sciences of Ukraine, Petropavlovskaya Str. 58, Sumy 40000, Ukraine. Tel.: +38 0542333089, +38 0965951202. E-mail address: [email protected] (A.A. Yanovska). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.011
natural polymer used for its antimicrobial properties [6] and film forming ability [7]. Several mechanisms for antibacterial activity of chitosan have been reported [8]: 1) positively charged chitosan reacts with negatively charged molecules at the cell surface changing the cell permeability and resulting in leakage of intracellular material to the media [9,10]; 2) chitosan binds to DNA and inhibits RNA synthesis [8,11]. Some authors reported that the antibacterial action of chitosan is a combination of both mechanisms involving changes in the hydrophilicity and charge density of the cell surface as well as changes in the characteristics of chitosan adsorption to the cell wall [8,12]. Owing to the high content of both amino and hydroxyl groups in its monomers, chitosan can easily form chelate complexes with metals [13,14]. The ability of chitosan to form complexes with silver ions originates from NH− 2 groups present in the b-(1-4)-glucosamine units of the polymer [15]. The chitosan pKa has been reported to be in the range between 6 and 7, so it has pH-responsive transition from a soluble to insoluble form near neutral pH [16]. Such physicochemical aspects of chitosan behavior are employed in the present work for film formation on Ti–6Al–4V substrates. Silver is known to have antibacterial activity inhibiting the growth of gram-positive and gram-negative bacteria [17,18], long-term activity [19] without the risk of bacterial resistance [20]. But it is reported that the lethal silver concentrations for human cell lines, i.e. human mesenchymal stem cells, lymphocytes, and monocytes, are in a range
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1–2.5 μg silver mL−1 [21]. Some authors suggested that silver concentrations higher than 10 μg mL− 1 can be toxic to some human cells [22,23]. Silver can be used as antimicrobial compound doped into a polymer composite film. The chitosan–Ag films proposed in [14] demonstrate an excellent antibacterial action against model bacteria, Escherichia coli. For this reason silver-containing materials have attracted a great interest in recent years. The most silver-containing antimicrobial biomaterials include either elemental silver or Ag+ (silver salts or silver complexes) incorporated into organic (polymers) or inorganic (bio glasses and HA) matrices [18]. Ag-loaded HA composites can be obtained by ionexchange (sol–gel or co-precipitation) and co-sputtering of silver and hydroxyapatite [18–20,24–28]. In this paper we describe the use of a thermal substrate method [29] for deposition of antibacterial coatings in aqueous solutions. The advantages of this method are the low deposition temperatures that allow introduction of biomolecules into coating material, deposition from aqueous solution; capability of formation HA–Ag coatings with various coating morphology; phase composition; and addition of antimicrobial compounds and drugs. In this work we propose a new effective technique for deposition HA–Ag and HA–Ag–CS coatings with various silver concentrations and antimicrobial activity. Experimental conditions when silver is co-deposited with HA were found. The emphasis has been made on the formation of composite HA–Ag and HA–CS–Ag coatings onto surfaces modified by a chitosan film and unmodified Ti–6Al–4V substrates. Discussion is given to the properties of deposited HA–Ag and HA–CS–Ag coatings (morphology, phase composition, adhesion to the substrate surface and antimicrobial activity), as well as to the influence of substrate surface pretreatment by chitosan film on the coating formation and silver content. 2. Materials and methods 2.1. Materials Ti–6Al–4 V specimens, 36 × 1.9 × 0.36 mm in size, were used as substrates for deposition of HA–CS–Ag and HA–Ag coatings. They were grinded with sandpaper (P100 grit), washed in acetone (15 min) and 96% ethanol (15 min) three times and rinsed with distilled water under ultrasound and finally dried in the air at room temperature. Biomedical grade chitosan (200 kDa molecular weight) was supplied by Haidebei Marine Bio Ltd. (Jinan, China) with 91% degree of deacetylation. Сa(NO3)2·4H2O, Na2HPO4·12H2O, NaOH, and CH3COOH (Sigma-Aldrich, Germany) were used. A 1 g L−1 chitosan solution was prepared by dissolving 1 g of chitosan fibers in 1 L of 1% CH3COOH solution that has been stirred. A 5 g L−1 concentration of chitosan solution was prepared in a similar way. Deposition of hydroxyapatite was performed on a heated Ti-6Al–4 V substrate at temperature 90 °C from aqueous solution containing 2.36 g L−1 Сa(NO3)2·4H2O and 2.148 g L−1 Na2HPO4·12H2O. The pH of the solutions was adjusted to 6.65 by NaOH. Deposition with stirring was performed for 1 h. Chitosan solution with 1 g L− 1 concentration was mixed with the initial solution for HA synthesis consisted of Сa(NO3)2·4H2O
(2.36 g L−1)/Na2HPO4·12H2O (2.148 g L−1) in proportions as presented in Table 1. A 0.01 g L− 1 Ag+ concentration of solution was obtained by electrolysis with a soluble silver anode used. The chitosan film was obtained by dipping Ti–6Al–4V substrates into the 5 g L−1 chitosan in 1% CH3COOH solution, followed by immersion into a solution of NaOH (40 g L−1) and drying in the air for 48 h at room temperature. 2.2. Deposition of HA–CS–Ag and HA–Ag coatings by thermal substrate method The thermal substrate method used to form HA coatings is based on the principle that the solubility of HA in an aqueous solution decreases with increasing substrate temperature [30,31]. An alternating current is passed through the system to heat the substrate. By this method hydroxyapatite is directly deposited onto the substrate without precipitating in the initial solution. The experimental setup for deposition of coatings is presented in [29]. Hydroxyapatite coatings with chitosan and silver additions were deposited by thermal substrate method on substrates modified by a chitosan film and unmodified Ti–6Al–4V substrates at pH = 6.65, t°substrate = 90 °C, and a 1 hour treatment. The composition of the initial solutions for deposition of coatings is shown in Table 1. 2.3. Analysis techniques The crystallinity and structure of the coatings were examined using an X-ray diffractometer DRON 4-07 (“Burevestnik”, Russia) connected to a computer-aided system for experiment control and data processing. The Ni-filtered CuKα radiation (wavelength 0.154 nm) with a conventional Bragg–Brentano θ–2θ geometry was used. The current and the voltage of the X-ray tube were 20 mA and 40 kV, respectively. The samples were measured in the continuous mode at a rate of 1.0°/min, with 2θ-angles ranging from 15° to 55°. All experimental data were processed by the program package DIFWIN-1 (“Etalon PTC” Ltd, Russia). Identification of crystal phases was performed with reference to a JCPDS card catalog. The surface morphology of HA–CS–Ag and HA–Ag coatings was examined by scanning electron microscopy (SEM). These investigations were performed in combination with X-ray emission spectroscopy using the REMMA-102 device (SELMI, Sumy, Ukraine). The surface chemical composition was determined with an energy dispersive X-ray (EDX) detector. The analytical signal of the characteristic X-ray emission was integrated by scanning the 50 × 50 μm2 area of the sample surface. The Ag+ concentrations in the HA–Ag coatings were determined by atomic absorption spectrometry (AAS) using a CAS-120.1 device (SELMI, Sumy, Ukraine). Titanium substrates coated with hydroxyapatite were treated with 63% nitric acid to dissolve the coating. The volume of the sample was brought to 3 mL. The experimental conditions were: λ (Ag) = 328.1 nm and spectral slit width = 0.4 nm. Calibration solutions were prepared by diluting the standard sample solutions of metal salts (GSO 6077-91) to 0.25 and 0.5 mg L− 1 concentrations. The Ag
Table 1 The composition of initial solutions. Coating composition
HA–Ag (ms) (a) HA–Ag (ums) (b) HA–CS–Ag (ms) (c) HA–CS–Ag (ums) (d) HA (e)
Composition of initial solutions (vol./vol.) Chitosan solution in 1% CH3COOH, (1 g L−1)
Solution of Ag+ (0.01 g L−1)
Solution for HA synthesis 10 Сa(NO3)2·4H2O/6Na2HPO4·12H2O
– – 50 mL 50 mL –
50 mL 50 mL 50 mL 50 mL –
150 mL 150 mL 100 mL 100 mL 200 mL
(ms) —modified by chitosan film substrate, (ums) —unmodified substrate.
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concentration was calculated relative to the dry weight of hydroxyapatite coating in each sample. Adhesive strength of obtained coatings was measured by tape-test method. It was evaluated by examining whether separation of the HA–Ag and HA–CS–Ag coatings occurred when the tape was peeled off. Optical density of the culture medium E. coli containing experimental samples was measured by spectrophotometric method (repeated three times) using a SF-56 spectrophotometer (λ = 540 nm) with mathematical processing of obtained data. This method is based on measure of light-absorbing properties of cell cultures. Optical density of bacteria is determined by light scattering effect that is in direct proportion to the concentration of bacteria cells in the media. 2.4. Bacteria studies In this study a reference strain of E. coli ATCC 25922 (Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine) was used as a model to evaluate in vitro the antimicrobial properties of HA coatings with CS and Ag additions. Experimental samples were immersed into the sterilized tubes, containing 4 mL of physiological solution saline (0.9% NaCl), followed by incubation for 24 h at 37 °C in an oven to simulate physiological solution conditions. Thereafter 0.4 mL of the bacteria E. coli suspension with a concentration of 1.5 · 108 (CFU mL−1) was added into the tubes containing experimental samples to make the final concentration of the bacteria cells approximately 1.0 · 108 (CFU mL−1) for simulation of an inflammatory process infection. The bacterial suspension was prepared using a Densi-La-Meter (PLIVA-Lachema, the Czech Republic, λ = 540 nm) according to a standard procedure [32]. Optical density of the bacterial solutions was spectrophotometrically measured after 2, 24, and 48 h. Bacteria cell amount was evaluated from the optical density measurements of the bacteria suspension lines with known concentrations of CFU mL−1. In the present study the bacterial tests were triplicated. The mean values and standard deviations were calculated from the obtained data. HA-coated substrates without silver were used as reference samples. The bacterial cell concentration was calculated with reference to standard suspensions of known concentrations (CFU mL−1). Experiments were carried out in triplicate and results were expressed as mean values with standard deviations. We also investigated the ability of the E. coli to adhere to sheep erythrocytes, as previously described [33], under the influence of HA–Ag coatings. In brief, 1 mL of a suspension of E. coli (109 CFU mL−1) and 0.5 mL of erythrocytes suspension (108 cells/mL) were added to the tubes with samples and incubated for 30 min while being shaken at 37 °C. On a glass slide the investigated suspension was dried at room temperature, fixed with ethanol, colored and examined under a light optical microscope at ×100 magnification. A number of bacteria adherent to 50 erythrocytes were counted (three series of each sample) and mean values were calculated. Adhesive properties of bacteria were estimated by adhesive index (AI) calculated as follows: AI ¼
MVA 100 ; PCE
ð1Þ
chemical and phase composition of the coatings as well as coatings with addition of biomolecules. Silver doping of the coating material is possible in both cases by co-deposition from aqueous solutions. Since silver occurs in such solutions in the form of Ag+ ions, chitosan molecules form complexes due to the coordination interaction of its amino-groups with silver ions (Fig. 1). The presence of amino groups enables CS to exist in a soluble or solid form depending on the solution pH value. In the acetic acid solution amino-groups are protonated (CS–NH+ 3 ). Neutralized chitosan in the hydrogel form is protonated and characterized by a higher sorption capacity to silver ions in aqueous solutions [14]. Binding of cations is possible only after deprotonation of the amino-groups and it goes in two stages: at pH lower than 5.3, binding of cations with glucosamine molecules proceeds according to the “hanging droplet” model (the metal ion is bound with one amino group); while at pH above 5.8, it goes under the “bridge” model (the metal ions are bond with several amino groups of the same chain or of different chains) [14] (Fig. 1). The coating composition was varied by changing the HA, Ag and chitosan concentrations in the initial solutions, as presented in Table 1. The morphology of the HA–Ag and HA–CS–Ag coatings deposited on the modified (Fig. 2a, c) and unmodified (Fig. 2b, d) Ti–6Al–4V substrates are shown in Fig. 2. All coatings are deposited by the similar experimental procedure and have nearly the same chemical composition, thus their morphology is close to each other, but some differences were found. The EDX spectra show that intensities of Ti-peaks for the unmodified substrate coating are higher (Fig. 2b) than those for the modified substrate coatings (Fig. 2a). It is observed that Ti-peaks intensities are lower for HA–CS– Ag coatings deposited on the unmodified substrates, or absent (Fig. 2 c) for HA–CS–Ag coatings on the modified substrates (Fig. 2 d). We suggest that the coatings are uniform and thicker, as a substrate material is not observed from EDX spectra (Fig. 2c), in contrast to a coating deposited on the unmodified surface (Fig. 2d). The thickness of the coatings was measured with a micrometer SGM-Filetta and confirmed by SEM (substrate profile). The HA–Ag coating obtained on the modified substrate (Fig. 2 a) was uniform, with a thickness of about 120 μm, while for the HA–Ag coating on the unmodified substrate the thickness was nearly 50 μm. Similarly, the HA–CS–Ag coating thickness was also nearly twice (210 μm) that of the coating obtained on the unmodified substrate (120 μm). The modification of titanium substrates by a chitosan film is intended to improve the uniformity of the coatings and their adhesion to a substrate surface. The adhesive strength of such coatings depends mainly on the cohesion between the individual particles of the coating. On the adhesion testing, coating failure proceeds mainly via a cohesive way, especially for HA coating without chitosan addition, rather than adhesive one (at the implant-coating interface). The upper layer of the coating adhered to the tape and the lower layer adhered to the substrate in all cases. Adhesive strength was calculated to be around 12 MPa for the HA–Ag coating deposited on the unmodified substrate, instead of 14 MPa for HA–Ag coating on the substrate modified by a chitosan film. The value of adhesive strength for HA–CS–Ag coating (ums) is 16 MPa instead of 19 MPa for HA–CS–Ag coating (ums).
where AI is the adhesive index, a mean value of bacteria cells adhered to 1 erythrocyte; MVA is the mean value of adhesion, a mean value of bacteria cells that adhered to 1 erythrocyte at calculation of 50 erythrocytes; and PCE is the participation coefficient of erythrocytes, the percentage of erythrocytes with adhered bacteria on their surface. 3. Results and discussion HA–CS–Ag and HA–Ag coatings were deposited on the CS-modified and unmodified substrates by a thermal substrate method, which is a low temperature deposition process. It permits control over the
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Fig. 1. Scheme of chitosan–silver interaction.
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Fig. 2. Morphology of hydroxyapatite-based coatings deposited by thermal substrate method from aqueous solutions with varied composition: a) HA–Ag coating (ms), b) HA–Ag coating (ums), c) HA–CS–Ag coating (ms), and d) HA–CS–Ag coating (ums) where (ms)—modified substrate and (ums)—unmodified substrate.
A chitosan film creates crystallization centers for HA deposition due to complexes formed between amino-groups of chitosan and phosphate ions in aqueous solutions as described in literature [34–36]. It was reported that chitosan (in concentrations 20–70 mg L−1) added to the initial solution has the ability to effectively modulate HA crystallization. It may be explained by phosphorylation of chitosan molecules that interact with calcium phosphate precursor compounds [34]. Chitosan interacts with inorganic ions owing to its hydrophilic and reactive nature in aqueous electrolyte solutions [35]. Besides it is known for its chelation properties (interaction with Ca2+), but it has greater affinity for PO3− ions [36]. 4 Chitosan has pH-responsive transition from a soluble to insoluble form at near neutral pH in the range between 6 and 7 [16]. In the initial solution the components for hydroxyapatite synthesis and chitosan are in soluble state. Hydroxyapatite–chitosan coating is deposited as solution pH is increased up to 6.5–7 by adding of NaOH and heating of substrate to 80–100 °C. The substrate is heated with alternating current. It results in local increase in ions (Ca2 +, PO34 −, etc.) concentrations at a substrate surface, and coating deposition due to decrease in
hydroxyapatite solubility. According to XRD patterns (Fig. 3), the main phase of deposited coatings is hydroxyapatite (JCPDS 9-432). As observed in the XRD-analysis of obtained coatings, the lack of Ag peaks in diffraction patterns is attributed to the low concentration of Ag used in the coatings. The most intensive peak of Ag (111) is not evident at 2θ = 38.4° that indicates the absence of crystal phase of silver. Silver can be incorporated into the hydroxyapatite coatings in the form of ions (Ag+). XRD analysis was made for samples placed in a Plexiglas cuvette. In the XRD spectra the peak between 2θ = 20–25° corresponds to Plexiglas. Besides, chitosan main peak is also within this region. So, the peak of chitosan may be superimposed on the peak of Plexiglas. The silver concentration in the deposited coating was measured by AAS technique (Table 2). The amount of silver was roughly comparable for coatings deposited on the unmodified and modified substrates. It slightly increases when the HA–Ag coatings are deposited on the unmodified substrates. When HA–Ag coating immersed into physiological solution, silver release take place. The mechanism of silver interaction with bacteria cells is described in works [3,23,37]. Antibacterial action of silver is a result of binding of Ag+ ions with electron donor groups (sulfur, oxygen and nitrogen) in biomolecules which are present in microbial cells as amines, hydroxyls, phosphates, and thiols due to the complexation. Sulfhydryl groups of enzymes are inhibited by binding with Ag+, and it causes conformational change and thereby inhibits the electron transport chain of a cell [3]. Indirect toxicity may arise from salt formation with silver ions that results in a chloride or anion limitation within a cell [37]. Besides, addition of Ag+ ions affects biological functions (permeability and respiration) by interacting with a bacterial membrane. They also penetrate into the cell and cause damage as they bind with DNA and RNA and thereby prevent bacterial reproduction [3,38].
Table 2 AAS data for the silver content in HA–Ag coatings.
Fig. 3. X-ray diffraction patterns of the coatings deposited from aqueous solutions: a) HA–Ag coating (ms), b) HA–Ag coating (ums), c) HA–CS–Ag coating (ms), d) HA–CS– Ag coating (ums) and e) HA coating.
a b a
Composition of the initial solution for coating deposition
Concentration mg/g
Relative root-mean-square deviation, %
HA–Ag coating (ms)a HA–Ag coating (ums)a
4.19 4.75
11 4
(ms) —modified by chitosan film substrate, (ums) — unmodified substrate.
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be used for development of coatings for implant protection against infection.
Acknowledgments The authors wish to thank Osolodchenko T.P. (Mechnikov Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine) for assistance in microbiological investigations.
References
Fig. 4. Cell amount of bacterial suspensions (E. coli) after immersion of HA–CS (ms), HA–Ag (ums) and HA–Ag (ms) coatings in comparison with the HA coating as reference sample.
The antibacterial activity of the HA–Ag coating on the unmodified and modified substrates was examined using spectrophotometry by measuring the optical density of the culture medium containing the experimental samples. Hydroxyapatite coatings without Ag addition were taken as reference samples. In Fig. 4 it can be seen that both the HA–Ag (unmodified) and HA–Ag (modified) coatings have bacteriostatic effect on the strain of E. coli that is somewhat greater for the latter. As it is evident from Fig. 4, the amount of bacteria grown on the HA-coated substrate is greater than that for HA–Ag coatings, since the HA–Ag coated substrates contain silver as antimicrobial compound. For HA–Ag (a modified substrate) coatings after a 24- and a 48-hour immersion in the solution containing E. coli strain, the antibacterial effect is increased, which is supposedly caused by a co-action of chitosan and silver. Measurements of the adhesion of E. coli to the sheep erythrocytes in the presence of HA–Ag coatings show a decreasing adhesion index to 14% as compared to that of the reference sample (Table 3). This is explained by the influence of an antimicrobial compound (silver ions, released from a coating) on adhesive receptors of the bacteria. Future studies on Ag-doped HA and HA–CS coatings will concern their ability to induce bone formation in vivo, their stability, and silver release in a physiological solution.
4. Conclusions Surface modification by a chitosan film is proposed for pre-treatment of Ti–6Al–4V substrates to promote nucleation of HA–Ag composite coatings deposited by the thermal substrate method. The HA–Ag and HA–CS–Ag antibacterial coatings were formed by co-deposition in aqueous solutions. It is concluded that the HA–Ag (modified) and HA–CS–Ag coatings obtained via the thermal substrate method exhibited better cohesion between HA-crystals due to the organic component of the coatings and better adhesive strength to the substrate surface. The HA–Ag coatings deposited on modified and unmodified Ti–6Al–4V substrates exhibit the antibacterial activity against infection strain of E. coli as compared with the HA coating without Ag, and can
Table 3 Adhesive indexes of E. coli in the presence of examined samples. Sample
Adhesive Index of E. coli ATCC 25922
% to reference sample
HA (control) HA–Ag HA–Ag–CS
3.54 ± 0.01 3.06 ± 0.07 2.8 ± 0.01
100 86.4 79
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Silver-doped hydroxyapatite coatings formed on Ti–6Al–4V substrates and their characterization A.A. Yanovska a,⁎, A.S. Stanislavov a, L.B. Sukhodub c, V.N. Kuznetsov a, V.Yu. Illiashenko a, S.N. Danilchenko a, L.F. Sukhodub b a b c
Institute of Applied Physics National Academy of Sciences of Ukraine, 58, Sumy 40000, Ukraine Sumy State University, Medical Institute, Ministry of Education and Science of Ukraine, R. Korsakova Str. 2, Sumy 40007, Ukraine Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine, 14-Puschinskaya St., Kharkov 61057, Ukraine
a r t i c l e
i n f o
Article history: Received 28 September 2013 Received in revised form 4 November 2013 Accepted 6 December 2013 Available online 16 December 2013 Keywords: Hydroxyapatite (HA) Chitosan (CS) Silver Antibacterial Coating
a b s t r a c t Coatings with antibacterial components for medical implants are recommended to reduce the risk of bacterial infections. Therefore hydroxyapatite (HA) coatings with addition of chitosan (CS) and silver (Ag) are proposed in this work in an attempt to resolve this problem. Ti–6Al–4V substrates were modified by a chitosan film to study the influence of surface modification on the formation of the HA–Ag and HA–CS–Ag coatings. Using a thermal substrate method, HA and HA–CS coatings doped with Ag+ were prepared at low substrate temperatures (90 °C). Coated surfaces were examined using X-ray diffraction and scanning electron microscopy. The amount of silver in the deposited coatings was analyzed by atomic absorption spectroscopy. From this study it is concluded that the substrate surface modified by a chitosan film promotes the coating formation and increases the antibacterial activity of produced coatings against a strain of Escherichia coli. The adhesion of E. coli (ATCC 25922) to sheep erythrocytes was decreased by 14% as compared with the reference samples without Ag. It could be explained by the inhibition of bacterial adhesins by Ag+ ions released. The combined action of silver ions and chitosan resulted in a 21% decrease in adhesive index. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The problem of infections related to biomedical devices and implants still remains open in spite of active research in this field [1]. Bacterial infections associated with implant materials lead to the inflammation around the implant, eventually resulting in implant loss. Bacteria may readily colonize the surfaces of synthetic medical implants [1]. The implant-associated infections may be prevented by application of antibacterial coatings based on biopolymers and inorganic compounds. Because of excellent osteoconductivity and bioactivity, hydroxyapatite (HA) is one of the most widely used biomaterials for bone reconstruction [2], as a coating material for implants to improve their biological properties [3]. Chitosan (CS) is a biopolymer produced from the deacetylation of chitin, a copolymer consisting of N-acetyl-2-amino-2-deoxy-Dglucopyranose and 2-amino-2-deoxy-D-glucopyranose, where the two types of recurring units are linked by (1 → 4)-β-glycosidic bonds [4]. Chitin and chitosan are drawing increasingly greater attention due to their biological and physicochemical properties [5]. Chitosan is the ⁎ Corresponding author at: Institute of Applied Physics National Academy of Sciences of Ukraine, Petropavlovskaya Str. 58, Sumy 40000, Ukraine. Tel.: +38 0542333089, +38 0965951202. E-mail address: [email protected] (A.A. Yanovska). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.011
natural polymer used for its antimicrobial properties [6] and film forming ability [7]. Several mechanisms for antibacterial activity of chitosan have been reported [8]: 1) positively charged chitosan reacts with negatively charged molecules at the cell surface changing the cell permeability and resulting in leakage of intracellular material to the media [9,10]; 2) chitosan binds to DNA and inhibits RNA synthesis [8,11]. Some authors reported that the antibacterial action of chitosan is a combination of both mechanisms involving changes in the hydrophilicity and charge density of the cell surface as well as changes in the characteristics of chitosan adsorption to the cell wall [8,12]. Owing to the high content of both amino and hydroxyl groups in its monomers, chitosan can easily form chelate complexes with metals [13,14]. The ability of chitosan to form complexes with silver ions originates from NH− 2 groups present in the b-(1-4)-glucosamine units of the polymer [15]. The chitosan pKa has been reported to be in the range between 6 and 7, so it has pH-responsive transition from a soluble to insoluble form near neutral pH [16]. Such physicochemical aspects of chitosan behavior are employed in the present work for film formation on Ti–6Al–4V substrates. Silver is known to have antibacterial activity inhibiting the growth of gram-positive and gram-negative bacteria [17,18], long-term activity [19] without the risk of bacterial resistance [20]. But it is reported that the lethal silver concentrations for human cell lines, i.e. human mesenchymal stem cells, lymphocytes, and monocytes, are in a range
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1–2.5 μg silver mL−1 [21]. Some authors suggested that silver concentrations higher than 10 μg mL− 1 can be toxic to some human cells [22,23]. Silver can be used as antimicrobial compound doped into a polymer composite film. The chitosan–Ag films proposed in [14] demonstrate an excellent antibacterial action against model bacteria, Escherichia coli. For this reason silver-containing materials have attracted a great interest in recent years. The most silver-containing antimicrobial biomaterials include either elemental silver or Ag+ (silver salts or silver complexes) incorporated into organic (polymers) or inorganic (bio glasses and HA) matrices [18]. Ag-loaded HA composites can be obtained by ionexchange (sol–gel or co-precipitation) and co-sputtering of silver and hydroxyapatite [18–20,24–28]. In this paper we describe the use of a thermal substrate method [29] for deposition of antibacterial coatings in aqueous solutions. The advantages of this method are the low deposition temperatures that allow introduction of biomolecules into coating material, deposition from aqueous solution; capability of formation HA–Ag coatings with various coating morphology; phase composition; and addition of antimicrobial compounds and drugs. In this work we propose a new effective technique for deposition HA–Ag and HA–Ag–CS coatings with various silver concentrations and antimicrobial activity. Experimental conditions when silver is co-deposited with HA were found. The emphasis has been made on the formation of composite HA–Ag and HA–CS–Ag coatings onto surfaces modified by a chitosan film and unmodified Ti–6Al–4V substrates. Discussion is given to the properties of deposited HA–Ag and HA–CS–Ag coatings (morphology, phase composition, adhesion to the substrate surface and antimicrobial activity), as well as to the influence of substrate surface pretreatment by chitosan film on the coating formation and silver content. 2. Materials and methods 2.1. Materials Ti–6Al–4 V specimens, 36 × 1.9 × 0.36 mm in size, were used as substrates for deposition of HA–CS–Ag and HA–Ag coatings. They were grinded with sandpaper (P100 grit), washed in acetone (15 min) and 96% ethanol (15 min) three times and rinsed with distilled water under ultrasound and finally dried in the air at room temperature. Biomedical grade chitosan (200 kDa molecular weight) was supplied by Haidebei Marine Bio Ltd. (Jinan, China) with 91% degree of deacetylation. Сa(NO3)2·4H2O, Na2HPO4·12H2O, NaOH, and CH3COOH (Sigma-Aldrich, Germany) were used. A 1 g L−1 chitosan solution was prepared by dissolving 1 g of chitosan fibers in 1 L of 1% CH3COOH solution that has been stirred. A 5 g L−1 concentration of chitosan solution was prepared in a similar way. Deposition of hydroxyapatite was performed on a heated Ti-6Al–4 V substrate at temperature 90 °C from aqueous solution containing 2.36 g L−1 Сa(NO3)2·4H2O and 2.148 g L−1 Na2HPO4·12H2O. The pH of the solutions was adjusted to 6.65 by NaOH. Deposition with stirring was performed for 1 h. Chitosan solution with 1 g L− 1 concentration was mixed with the initial solution for HA synthesis consisted of Сa(NO3)2·4H2O
(2.36 g L−1)/Na2HPO4·12H2O (2.148 g L−1) in proportions as presented in Table 1. A 0.01 g L− 1 Ag+ concentration of solution was obtained by electrolysis with a soluble silver anode used. The chitosan film was obtained by dipping Ti–6Al–4V substrates into the 5 g L−1 chitosan in 1% CH3COOH solution, followed by immersion into a solution of NaOH (40 g L−1) and drying in the air for 48 h at room temperature. 2.2. Deposition of HA–CS–Ag and HA–Ag coatings by thermal substrate method The thermal substrate method used to form HA coatings is based on the principle that the solubility of HA in an aqueous solution decreases with increasing substrate temperature [30,31]. An alternating current is passed through the system to heat the substrate. By this method hydroxyapatite is directly deposited onto the substrate without precipitating in the initial solution. The experimental setup for deposition of coatings is presented in [29]. Hydroxyapatite coatings with chitosan and silver additions were deposited by thermal substrate method on substrates modified by a chitosan film and unmodified Ti–6Al–4V substrates at pH = 6.65, t°substrate = 90 °C, and a 1 hour treatment. The composition of the initial solutions for deposition of coatings is shown in Table 1. 2.3. Analysis techniques The crystallinity and structure of the coatings were examined using an X-ray diffractometer DRON 4-07 (“Burevestnik”, Russia) connected to a computer-aided system for experiment control and data processing. The Ni-filtered CuKα radiation (wavelength 0.154 nm) with a conventional Bragg–Brentano θ–2θ geometry was used. The current and the voltage of the X-ray tube were 20 mA and 40 kV, respectively. The samples were measured in the continuous mode at a rate of 1.0°/min, with 2θ-angles ranging from 15° to 55°. All experimental data were processed by the program package DIFWIN-1 (“Etalon PTC” Ltd, Russia). Identification of crystal phases was performed with reference to a JCPDS card catalog. The surface morphology of HA–CS–Ag and HA–Ag coatings was examined by scanning electron microscopy (SEM). These investigations were performed in combination with X-ray emission spectroscopy using the REMMA-102 device (SELMI, Sumy, Ukraine). The surface chemical composition was determined with an energy dispersive X-ray (EDX) detector. The analytical signal of the characteristic X-ray emission was integrated by scanning the 50 × 50 μm2 area of the sample surface. The Ag+ concentrations in the HA–Ag coatings were determined by atomic absorption spectrometry (AAS) using a CAS-120.1 device (SELMI, Sumy, Ukraine). Titanium substrates coated with hydroxyapatite were treated with 63% nitric acid to dissolve the coating. The volume of the sample was brought to 3 mL. The experimental conditions were: λ (Ag) = 328.1 nm and spectral slit width = 0.4 nm. Calibration solutions were prepared by diluting the standard sample solutions of metal salts (GSO 6077-91) to 0.25 and 0.5 mg L− 1 concentrations. The Ag
Table 1 The composition of initial solutions. Coating composition
HA–Ag (ms) (a) HA–Ag (ums) (b) HA–CS–Ag (ms) (c) HA–CS–Ag (ums) (d) HA (e)
Composition of initial solutions (vol./vol.) Chitosan solution in 1% CH3COOH, (1 g L−1)
Solution of Ag+ (0.01 g L−1)
Solution for HA synthesis 10 Сa(NO3)2·4H2O/6Na2HPO4·12H2O
– – 50 mL 50 mL –
50 mL 50 mL 50 mL 50 mL –
150 mL 150 mL 100 mL 100 mL 200 mL
(ms) —modified by chitosan film substrate, (ums) —unmodified substrate.
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concentration was calculated relative to the dry weight of hydroxyapatite coating in each sample. Adhesive strength of obtained coatings was measured by tape-test method. It was evaluated by examining whether separation of the HA–Ag and HA–CS–Ag coatings occurred when the tape was peeled off. Optical density of the culture medium E. coli containing experimental samples was measured by spectrophotometric method (repeated three times) using a SF-56 spectrophotometer (λ = 540 nm) with mathematical processing of obtained data. This method is based on measure of light-absorbing properties of cell cultures. Optical density of bacteria is determined by light scattering effect that is in direct proportion to the concentration of bacteria cells in the media. 2.4. Bacteria studies In this study a reference strain of E. coli ATCC 25922 (Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine) was used as a model to evaluate in vitro the antimicrobial properties of HA coatings with CS and Ag additions. Experimental samples were immersed into the sterilized tubes, containing 4 mL of physiological solution saline (0.9% NaCl), followed by incubation for 24 h at 37 °C in an oven to simulate physiological solution conditions. Thereafter 0.4 mL of the bacteria E. coli suspension with a concentration of 1.5 · 108 (CFU mL−1) was added into the tubes containing experimental samples to make the final concentration of the bacteria cells approximately 1.0 · 108 (CFU mL−1) for simulation of an inflammatory process infection. The bacterial suspension was prepared using a Densi-La-Meter (PLIVA-Lachema, the Czech Republic, λ = 540 nm) according to a standard procedure [32]. Optical density of the bacterial solutions was spectrophotometrically measured after 2, 24, and 48 h. Bacteria cell amount was evaluated from the optical density measurements of the bacteria suspension lines with known concentrations of CFU mL−1. In the present study the bacterial tests were triplicated. The mean values and standard deviations were calculated from the obtained data. HA-coated substrates without silver were used as reference samples. The bacterial cell concentration was calculated with reference to standard suspensions of known concentrations (CFU mL−1). Experiments were carried out in triplicate and results were expressed as mean values with standard deviations. We also investigated the ability of the E. coli to adhere to sheep erythrocytes, as previously described [33], under the influence of HA–Ag coatings. In brief, 1 mL of a suspension of E. coli (109 CFU mL−1) and 0.5 mL of erythrocytes suspension (108 cells/mL) were added to the tubes with samples and incubated for 30 min while being shaken at 37 °C. On a glass slide the investigated suspension was dried at room temperature, fixed with ethanol, colored and examined under a light optical microscope at ×100 magnification. A number of bacteria adherent to 50 erythrocytes were counted (three series of each sample) and mean values were calculated. Adhesive properties of bacteria were estimated by adhesive index (AI) calculated as follows: AI ¼
MVA 100 ; PCE
ð1Þ
chemical and phase composition of the coatings as well as coatings with addition of biomolecules. Silver doping of the coating material is possible in both cases by co-deposition from aqueous solutions. Since silver occurs in such solutions in the form of Ag+ ions, chitosan molecules form complexes due to the coordination interaction of its amino-groups with silver ions (Fig. 1). The presence of amino groups enables CS to exist in a soluble or solid form depending on the solution pH value. In the acetic acid solution amino-groups are protonated (CS–NH+ 3 ). Neutralized chitosan in the hydrogel form is protonated and characterized by a higher sorption capacity to silver ions in aqueous solutions [14]. Binding of cations is possible only after deprotonation of the amino-groups and it goes in two stages: at pH lower than 5.3, binding of cations with glucosamine molecules proceeds according to the “hanging droplet” model (the metal ion is bound with one amino group); while at pH above 5.8, it goes under the “bridge” model (the metal ions are bond with several amino groups of the same chain or of different chains) [14] (Fig. 1). The coating composition was varied by changing the HA, Ag and chitosan concentrations in the initial solutions, as presented in Table 1. The morphology of the HA–Ag and HA–CS–Ag coatings deposited on the modified (Fig. 2a, c) and unmodified (Fig. 2b, d) Ti–6Al–4V substrates are shown in Fig. 2. All coatings are deposited by the similar experimental procedure and have nearly the same chemical composition, thus their morphology is close to each other, but some differences were found. The EDX spectra show that intensities of Ti-peaks for the unmodified substrate coating are higher (Fig. 2b) than those for the modified substrate coatings (Fig. 2a). It is observed that Ti-peaks intensities are lower for HA–CS– Ag coatings deposited on the unmodified substrates, or absent (Fig. 2 c) for HA–CS–Ag coatings on the modified substrates (Fig. 2 d). We suggest that the coatings are uniform and thicker, as a substrate material is not observed from EDX spectra (Fig. 2c), in contrast to a coating deposited on the unmodified surface (Fig. 2d). The thickness of the coatings was measured with a micrometer SGM-Filetta and confirmed by SEM (substrate profile). The HA–Ag coating obtained on the modified substrate (Fig. 2 a) was uniform, with a thickness of about 120 μm, while for the HA–Ag coating on the unmodified substrate the thickness was nearly 50 μm. Similarly, the HA–CS–Ag coating thickness was also nearly twice (210 μm) that of the coating obtained on the unmodified substrate (120 μm). The modification of titanium substrates by a chitosan film is intended to improve the uniformity of the coatings and their adhesion to a substrate surface. The adhesive strength of such coatings depends mainly on the cohesion between the individual particles of the coating. On the adhesion testing, coating failure proceeds mainly via a cohesive way, especially for HA coating without chitosan addition, rather than adhesive one (at the implant-coating interface). The upper layer of the coating adhered to the tape and the lower layer adhered to the substrate in all cases. Adhesive strength was calculated to be around 12 MPa for the HA–Ag coating deposited on the unmodified substrate, instead of 14 MPa for HA–Ag coating on the substrate modified by a chitosan film. The value of adhesive strength for HA–CS–Ag coating (ums) is 16 MPa instead of 19 MPa for HA–CS–Ag coating (ums).
where AI is the adhesive index, a mean value of bacteria cells adhered to 1 erythrocyte; MVA is the mean value of adhesion, a mean value of bacteria cells that adhered to 1 erythrocyte at calculation of 50 erythrocytes; and PCE is the participation coefficient of erythrocytes, the percentage of erythrocytes with adhered bacteria on their surface. 3. Results and discussion HA–CS–Ag and HA–Ag coatings were deposited on the CS-modified and unmodified substrates by a thermal substrate method, which is a low temperature deposition process. It permits control over the
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Fig. 1. Scheme of chitosan–silver interaction.
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Fig. 2. Morphology of hydroxyapatite-based coatings deposited by thermal substrate method from aqueous solutions with varied composition: a) HA–Ag coating (ms), b) HA–Ag coating (ums), c) HA–CS–Ag coating (ms), and d) HA–CS–Ag coating (ums) where (ms)—modified substrate and (ums)—unmodified substrate.
A chitosan film creates crystallization centers for HA deposition due to complexes formed between amino-groups of chitosan and phosphate ions in aqueous solutions as described in literature [34–36]. It was reported that chitosan (in concentrations 20–70 mg L−1) added to the initial solution has the ability to effectively modulate HA crystallization. It may be explained by phosphorylation of chitosan molecules that interact with calcium phosphate precursor compounds [34]. Chitosan interacts with inorganic ions owing to its hydrophilic and reactive nature in aqueous electrolyte solutions [35]. Besides it is known for its chelation properties (interaction with Ca2+), but it has greater affinity for PO3− ions [36]. 4 Chitosan has pH-responsive transition from a soluble to insoluble form at near neutral pH in the range between 6 and 7 [16]. In the initial solution the components for hydroxyapatite synthesis and chitosan are in soluble state. Hydroxyapatite–chitosan coating is deposited as solution pH is increased up to 6.5–7 by adding of NaOH and heating of substrate to 80–100 °C. The substrate is heated with alternating current. It results in local increase in ions (Ca2 +, PO34 −, etc.) concentrations at a substrate surface, and coating deposition due to decrease in
hydroxyapatite solubility. According to XRD patterns (Fig. 3), the main phase of deposited coatings is hydroxyapatite (JCPDS 9-432). As observed in the XRD-analysis of obtained coatings, the lack of Ag peaks in diffraction patterns is attributed to the low concentration of Ag used in the coatings. The most intensive peak of Ag (111) is not evident at 2θ = 38.4° that indicates the absence of crystal phase of silver. Silver can be incorporated into the hydroxyapatite coatings in the form of ions (Ag+). XRD analysis was made for samples placed in a Plexiglas cuvette. In the XRD spectra the peak between 2θ = 20–25° corresponds to Plexiglas. Besides, chitosan main peak is also within this region. So, the peak of chitosan may be superimposed on the peak of Plexiglas. The silver concentration in the deposited coating was measured by AAS technique (Table 2). The amount of silver was roughly comparable for coatings deposited on the unmodified and modified substrates. It slightly increases when the HA–Ag coatings are deposited on the unmodified substrates. When HA–Ag coating immersed into physiological solution, silver release take place. The mechanism of silver interaction with bacteria cells is described in works [3,23,37]. Antibacterial action of silver is a result of binding of Ag+ ions with electron donor groups (sulfur, oxygen and nitrogen) in biomolecules which are present in microbial cells as amines, hydroxyls, phosphates, and thiols due to the complexation. Sulfhydryl groups of enzymes are inhibited by binding with Ag+, and it causes conformational change and thereby inhibits the electron transport chain of a cell [3]. Indirect toxicity may arise from salt formation with silver ions that results in a chloride or anion limitation within a cell [37]. Besides, addition of Ag+ ions affects biological functions (permeability and respiration) by interacting with a bacterial membrane. They also penetrate into the cell and cause damage as they bind with DNA and RNA and thereby prevent bacterial reproduction [3,38].
Table 2 AAS data for the silver content in HA–Ag coatings.
Fig. 3. X-ray diffraction patterns of the coatings deposited from aqueous solutions: a) HA–Ag coating (ms), b) HA–Ag coating (ums), c) HA–CS–Ag coating (ms), d) HA–CS– Ag coating (ums) and e) HA coating.
a b a
Composition of the initial solution for coating deposition
Concentration mg/g
Relative root-mean-square deviation, %
HA–Ag coating (ms)a HA–Ag coating (ums)a
4.19 4.75
11 4
(ms) —modified by chitosan film substrate, (ums) — unmodified substrate.
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be used for development of coatings for implant protection against infection.
Acknowledgments The authors wish to thank Osolodchenko T.P. (Mechnikov Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine) for assistance in microbiological investigations.
References
Fig. 4. Cell amount of bacterial suspensions (E. coli) after immersion of HA–CS (ms), HA–Ag (ums) and HA–Ag (ms) coatings in comparison with the HA coating as reference sample.
The antibacterial activity of the HA–Ag coating on the unmodified and modified substrates was examined using spectrophotometry by measuring the optical density of the culture medium containing the experimental samples. Hydroxyapatite coatings without Ag addition were taken as reference samples. In Fig. 4 it can be seen that both the HA–Ag (unmodified) and HA–Ag (modified) coatings have bacteriostatic effect on the strain of E. coli that is somewhat greater for the latter. As it is evident from Fig. 4, the amount of bacteria grown on the HA-coated substrate is greater than that for HA–Ag coatings, since the HA–Ag coated substrates contain silver as antimicrobial compound. For HA–Ag (a modified substrate) coatings after a 24- and a 48-hour immersion in the solution containing E. coli strain, the antibacterial effect is increased, which is supposedly caused by a co-action of chitosan and silver. Measurements of the adhesion of E. coli to the sheep erythrocytes in the presence of HA–Ag coatings show a decreasing adhesion index to 14% as compared to that of the reference sample (Table 3). This is explained by the influence of an antimicrobial compound (silver ions, released from a coating) on adhesive receptors of the bacteria. Future studies on Ag-doped HA and HA–CS coatings will concern their ability to induce bone formation in vivo, their stability, and silver release in a physiological solution.
4. Conclusions Surface modification by a chitosan film is proposed for pre-treatment of Ti–6Al–4V substrates to promote nucleation of HA–Ag composite coatings deposited by the thermal substrate method. The HA–Ag and HA–CS–Ag antibacterial coatings were formed by co-deposition in aqueous solutions. It is concluded that the HA–Ag (modified) and HA–CS–Ag coatings obtained via the thermal substrate method exhibited better cohesion between HA-crystals due to the organic component of the coatings and better adhesive strength to the substrate surface. The HA–Ag coatings deposited on modified and unmodified Ti–6Al–4V substrates exhibit the antibacterial activity against infection strain of E. coli as compared with the HA coating without Ag, and can
Table 3 Adhesive indexes of E. coli in the presence of examined samples. Sample
Adhesive Index of E. coli ATCC 25922
% to reference sample
HA (control) HA–Ag HA–Ag–CS
3.54 ± 0.01 3.06 ± 0.07 2.8 ± 0.01
100 86.4 79
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