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Antibacterial, cytotoxicity and physical properties of laser — Silver doped hydroxyapatite layers
Materials Science and Engineering C 33 (2013) 1242–1246
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Antibacterial, cytotoxicity and physical properties of laser — Silver doped hydroxyapatite layers M. Jelinek a, b,⁎, T. Kocourek a, b, J. Remsa a, b, M. Weiserová c, K. Jurek a, J. Mikšovský a, b, J. Strnad d, A. Galandáková e, J.Ulrichová e a
Institute of Physics ASCR v.v.i., Na Slovance 2, 18221 Prague 8, Czech Republic Czech Technical University in Prague, Faculty of Biomedical Engineering, Sitna 3105, Kladno, Czech Republic Institute of Microbiology ASCR v.v.i., Vídeňská 1083, Prague 4, Czech Republic d Lasak Ltd., Papirenská 25, 1600 Prague 6, Czech Republic e The Faculty of Medicine and Dentistry, Palacký University in Olomouc, Hněvotínská 3, 775 15 Olomouc, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 12 June 2012 Received in revised form 27 September 2012 Accepted 3 December 2012 Available online 9 December 2012 Keywords: Hydroxyapatite Silver Thin films PLD Antibacterial Cytotoxicity
a b s t r a c t Hydroxyapatite layers with silver doping from 0.06 at.% to 14 at.% were prepared by laser deposition. The films' physical properties such as morphology, composition, crystallinity, Young's modulus and microhardness were measured. Films were amorphous or polycrystalline in dependence on deposition temperature (from RT to 600 °C). Antibacterial properties were tested using Escherichia coli and Bacillus subtilis cells. The antibacterial efficacy changed with silver doping from 4% to 100%. Cytotoxicity was studied by a direct contact test. Depending on doping and crystallinity the films were either non-toxic or mildly toxic. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Metallic silver and silver compounds as hydroxyapatite (HA)–silver composites, are widely used in medical devices and health care products [1]. Besides the mechanical properties, antibacterial properties and cytotoxicity are also important for medical applications. Bacterial infections are usually caused by the adherence and colonization of bacteria on coated implants [2]. Silver and silver ions have long been known to have strong inhibitory and bactericidal effects as well as a broad spectrum of anti-microbial activities [2]. Antibacterial tests evaluate anti-bacterial performance of silver against bacteria. Different positive and negative strains were used in testing such as: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus [2], Staphylococcus epidermis [3], Streptococcus mutants [4] and Streptococcus pyogeneses [1]. Cytotoxicity is the capacity of an agent to be toxic to cells. Cytotoxicity test of silver doped hydroxyapatite was evaluated with fibroblast cell [2], human embryonic palatal mesenchymal cells [5], human osteoblast cell line [6], murine macrophages [7], agar overlay cytotoxic assay [8], C-reactive proteins, and neutrophilic leukocytes [9]. Various opinions concerning biocompatibility were presented in the ⁎ Corresponding author at: Institute of Physics ASCR v.v.i., Na Slovance 2, 18221 Prague 8, Czech Republic. Tel.: +420 266052733. E-mail address: [email protected] (M. Jelinek). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.12.018
literature: silver exerts its antimicrobial action at low concentrations (1 ppm) [1], no significant cytotoxicity was observed for low Ag concentration [3,6], silver toxicity is dose dependent [4] and can be toxic [8,10–12]. The conclusions published in the mentioned studies are different and hard to compare. The reason is that the properties of doped materials depend on the dopant concentration, shape of material (bulk, thin layer, nanoparticles) and on the methods and parameters used to introduce silver into the material. It is known that different forms of Ag confer different degrees of bactericidal properties [3]. Minimal level of silver ions necessary in any situation to clear infections is not known yet. Further studies are urgently required [1]. One of the frequently used method for fabrication of HA films is pulsed laser deposition (PLD) [13]. In our contribution we studied silver doped hydroxyapatite layers prepared by PLD. Films of a wide scale of silver doping were created. The physical and antibacterial properties and cytotoxicity are presented and discussed. 2. Materials and methods Thin films of silver, HA and Ag + HA were prepared by PLD using a KrF excimer laser (λ = 248 nm, τ = 20 ns, rep. rate of 10 Hz). The laser beam was focused on a silver, HA or Ag + HA target with energy density of 2 J cm −2. The HA target was pressed from HA powder
M. Jelinek et al. / Materials Science and Engineering C 33 (2013) 1242–1246
supplied by Merck Co. The angle between the target normal and laser radiation was 45°. The target was rotated. Substrate (titanium, fused silica or Si(111)) was in a distance of 4 cm from the target. The deposition conditions to grow HA, Ag+ HA and Ag layers was the same. Substrate was held at room temperature or heated up to 600 °C, to create amorphous or crystalline films. Films were grown in a mixture of water vapor and argon of 40 Pa (water — 22 Pa, Ar — 18 Pa). The layers' thickness was determined by Alpha-step IQ mechanical profilometer (KLA Co.). The composition (WDS — wavelength dependence X-ray spectrometer) was analyzed with EDAX Jeol Supersprobe 733. Jeol was also used for the study of morphology (SEM — scanning electron microscopy) measurements. Young's modulus and microhardness were tested by Hysitron Triboindenter® TI950 device with head NanoScratch transducer SN5-483-194 (Pmax = 10 mN). The testing probe was diamond of Berkovich shape. For indentation, quasistatic loads with partial unloads (to 50% of the load) were used, and the time of each phase (load, dwell and unloading time) was 5 s. Used force was from 1 to 5 mN with 1 mN step. On each sample, 9 points (in 3 × 3 matrix) of indents with distances of tested points 20 μm were arranged. 2.1. In vitro tests The antibacterial properties of Ag + HA films were tested on the Gram-negative laboratory strain E. coli K12 C600 [14] and on the Gram-negative strain B. subtilis 168, commonly found in soil [15]. Cells were grown in Lysogeny Broth (LB) [16] medium and plated on LA plates (LA; LB with addition of 15% of agar). Tested silver doped HA layers were placed into sterile glass chambers and covered with 1 ml of the overnight cultures diluted in the sterile physiological saline solution on the concentration of cells 10 6 CFU/ml. Glass chambers were cultivated in 37 °C and aliquots of 100 μl were withdrawn at the time points and serially diluted up to 10 −6 in physiological saline solution. 100 μl of each diluted sample were spread over an agar plate. The plates were incubated overnight and number of growing colonies was counted. The bactericidal efficacy of different silver doped HA layers was estimated according the formula [17]: bactericidal ef f icacyð% Þ ¼
alive number in ref erence group alive number in experimental group ⋅100: alive number in ref erence group
Cytotoxicity was evaluated in six samples, amorphous or polycrystalline, containing pure hydroxyapatite or silver doped hydroxyapatite (doping 1.2% or 4.4%). Cytotoxicity was evaluated using a biocompatibility direct contact test on the mouse Swiss NIH embryo cell line (NIH 3T3), obtained from European Collection of Cell Cultures (UK). The cells were grown in Dulbecco's modified Eagle's medium supplemented with heat-inactivated bovine serum (10%,v/v), streptomycin(100 U/ml) and penicillin (0.1 mg/ml). Cells were incubated in a humidified atmosphere with 5% (v/v) CO2 at 37 °C.
materials, similar shape titan sample (negative control) and copper sample (positive control) were used. After the incubation period the cultures were analyzed using an inversion microscope (Olympus, Japan). Inhibition of cell growth was quantified by taking the distance between the sample and the area where morphologically no differences were seen (decolonization index, lysis index) in comparison to control cultures. After that, the samples and medium were carefully removed, cells were fixed with methanol (1 min), colored with crystalline violet (2%, m/v in 20% methanol; 5 min), properly washed with water and dried. The decolonization index, lysis index and cell response were determined according to ISO 10993-5:2005 and ISO 7405:2008. Finally, the cell response to a test sample is based on the median decolonization index and lysis index of at least three replicate tests. 3. Results and discussion 3.1. Crystallinity The layers created at low substrate temperature were amorphous and the layers created at temperatures higher than 520 °C were polycrystalline [18,19]. Example of XRD of doped HA film is shown in Fig. 1 (only Ag and HA peaks were observed). 3.2. Composition From WDS measurement it follows that Ca/P ratio of our doped and undoped layers is rather higher than that for stoichiometric composition (1.67), but it is nearly unchanged for all silver doping (from zero doping to 14 at.% of Ag) — see Table 1. The result is different from that of [20], where it was observed that Ca/P decreased with an increasing Ag content. Because the ratio is unchanged, we can conclude that the bioresorbability is unchanged for our doped and undoped layers. We also measured the silver content in droplets: it was slightly lower or higher than that measured in smooth parts of layer. We confirmed that the content of droplets is a mix of silver and HA (there is no pure silver). 3.3. Morphology Layers were smoothly covered with small droplets. The presence of small silver content contributes to smoother layers — see Fig. 2. 3.4. Young's modulus (Y) and hardness (H) Young's modulus (Y) and hardness (H) of our amorphous HA and Ag + HA films are in the range from 45 GPa to 8 GPa, respectively from 0.3 GPa to 0.7 GPa (Fig. 3). There is a small change in Y and H
2.2. Biocompatibility direct contact test The direct contact biocompatibility test is based on the addition of a test sample directly on the top of a sub-confluent cell layer (without agar layer in between). The guidance for the test is in accordance with the international standards compiled as ISO 10993-5:2005 (Tests for cytotoxicity — In Vitro Methods) and ISO 7405:2008 (Dentistry — Evaluation of biocompatibility of medical devices used in dentistry).The cells were seeded in Petri dishes (6 cm diameter; 7 ml culture medium) at a density of 1 × 105 cells/ml and incubated in a humidified atmosphere with 5% (v/v) CO2 at 37 °C for 24 h. Then the culture medium was changed to the serum free one and the test sample controls were applied on the top of the cell layer and incubated for 24 h. As reference
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Fig. 1. XRD spectrum of HAAG4.4A layer (4.4 at.% of Ag).
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M. Jelinek et al. / Materials Science and Engineering C 33 (2013) 1242–1246
Table 1 Structure, WDX composition and bactericidal efficacy of HA and Ag + HA layers on fused silica substrates. Ca/P
Bacterial efficacy [%] Gram positive
Bacterial efficacy [%] Gram negative
0
1.9
–
–
–
1.2
–
–
–
–
–
4.4
–
99.9
Amorph.
21.9
13.6
8.3
1.6
–
100
Amorph.
–
–
–
–
100
Amorph.
–
–
100
–
–
99.2
Polycryst.
–
–
0
–
–
–
Polycryst.
26.1
13.6
0.06
1.9
71.0
3.9
Polycryst.
24.9
14.2
0.3
1.7
71.8
–
Polycryst.
25.8
13.4
1.2
1.9
93.4
99.9
Polycryst.
25.3
12.9
4.4
1.9
99.9
–
Polycryst.
22.2
11.5
13.7
1.9
99.9
100
Sample
HA structure
Ca [at.%]
P [at.%]
Ag [at.%]
HAAG 0.0A HAAG 1.2A HAAG 4.4A HAAG 8.3A HAAG 13.7A HAAG 100A HAAG 0.0C HAAG 0.06C HAAG 0.3C HAAG 1.2C HAAG 4.4C HAAG 13.7C
Amorph.
25.9
13.7
Amorph.
–
Amorph.
13.7
moduli with increase of silver content. For amorphous samples the Y and H moduli are significantly lower than that for crystalline samples. For polycrystalline samples the Y and H moduli are in the range from 141 GPa to 100 GPa, respectively from 6.8 GPa to 3.0 GPa (Fig. 3). In polycrystalline layers there is also a small change in Y modulus with silver concentration. As to hardness we observed a decrease of H with silver increase. The value of Y and H moduli depends on the substrate material (Fig. 3). The measured dependences are partly in agreement with [20] where the decrease of Y and H was observed with an increase of Ag concentration. From literature it follows that the Y and H moduli of pure HA are in the range from 85 GPa to 90 GPa and from 2.4 GPa to 5.9 GPa, respectively [20–23]. The Y and H moduli of silver are 82.7 GPa and 0.15 GPa–1.8 GPa, respectively [19]. We see that our measurements of amorphous layers show lower values and the results reached with polycrystalline layers show higher values than those we can find in the literature.
Fig. 3. Young's modulus (top) and hardness (below) for amorphous and crystalline Ag + HA samples prepared on silicon and titanium substrates.
3.5. In vitro tests HA layers doped with various concentration of silver (0.06%; 0.3%; 1.2%; 4.4%; 8.3% and 13.7%) and various structures were tested for their antimicrobial effect against the E. coli and B. subtilis strains. Data presented are an average of 5 measurements. 3.5.1. Gram-positive strain The number of B. subtilis colonies was reduced both in time and with a silver concentration. Except of layers with very low content of silver (0.06%) all analyzed tablets exhibited a bactericidal efficacy even after 1 h of cultivation with the bacteria. After 24 h of cultivation the bactericidal efficacy increased gradually to 99.9% (for all samples with doping of 4.4% Ag or higher). In summary, for silver
Fig. 2. SEM photo of HA layer (a — thickness of 300 nm), and Ag + HA layers (b — 4 at.% Ag, thickness of 500 nm, c — 13.7 at.% Ag, thickness of 800 nm). Magnification 400 ×. Maximum size of droplets: a) 8 μm, b) 5 μm, and c) 8 μm.
M. Jelinek et al. / Materials Science and Engineering C 33 (2013) 1242–1246 Table 2 Cytotoxicity of HA and doped Ag + HA layers. Sample
HAAG 0.0A HAAG 1.2A HAAG 4.4A HAAG 0.0C HAAG 1.2C HAAG 4.4C
Decolonization index/lysis index
Cytotoxicity Interpretation
0
0/0
0
Non-cytotoxic
0.83
1/1
1
Mildly toxic
Decolonization index [mm] I.
II.
III. Average
0
0
0
1
0.5 1
1.5 1
2
1.5
1/1
1
Mildly toxic
0
0
0
0
0/0
0
Non-cytotoxic
0
0
0
0
0/0
0
Non-cytotoxic
0
0
0
0
0/0
0
Non-cytotoxic
doping in region from 0.06% to 13.7% the antibacterial efficacy changed from 71% to 99.9% (Table 1). All tested layers were polycrystalline.
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3.5.2. Gram-negative strain Doped layers were tested for their antimicrobial effect against the strain E. coli C600. Comparison of their bactericidal efficacy expressed as percentage of killed cells is presented in Table 1. Except of layers with very low content of silver (0.06%Ag) all analyzed tablets exhibited excellent bactericidal efficacy (> 99.9%). Layers with silver concentration higher than 8.3% reached nearly 100% efficacy both for polycrystalline and amorphous layers. Cytotoxicity was evaluated in six samples, amorphous or polycrystalline, containing pure hydroxyapatite or silver doped hydroxyapatite (doping 1.2% or 4.4%). Neither visible alterations to NIH 3T3 monolayer nor cytotoxic effects (destruction of cells, decolonized zone, lysis cells) were found using inversion microscope and crystalline violet incorporation to amorphous and polycrystalline hydroxyapatite (HAAG0.0A and HAAG0.0C) and doped polycrystalline hydroxyapatite (HAAG1.2C and HAAG4.4C) samples. The results indicate that specimens were non-toxic (interpretation decolonization index/index lysis 0/0) and fully biocompatible with the cell model used in the study (Table 2, Fig. 4c–f). Mild cytotoxic effect (mild
Fig. 4. Effect of tested specimens on the NIH 3T3 cell line. a) Titan sample (negative control); b) cooper sample (positive control); c) HAAG0.0A; d) HAAG0.0C; e) HAAG1.2C; f) HAAG4.4C; g) HAAG1.2A and h) HAAG4.4A. The samples were observed at magnification 100 ×.
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M. Jelinek et al. / Materials Science and Engineering C 33 (2013) 1242–1246
destruction of cells, decolonization zone characteristic for lyses cell 1/1) was observed in doped amorphous (HAAG1.2A and HAAG4.4A) samples (Table 2, Fig. 4g and h). Our results of cytotoxicity test partly agree with conclusions in [2] where they found that for plasma sprayed silver doped HA coating the layers containing b5% of silver have no cytotoxic effect. 4. Conclusions The silver doped HA layers with doping of 0.06%, 0.3%, 1.2%, 4.4%, 8.3% and 13.7% were fabricated by PLD method. Depending on deposition conditions the films were amorphous or polycrystalline. We observed a distinct difference in Young's modulus Y and hardness H for amorphous and polycrystalline HA and Ag + HA films and the decrease in Y and H with silver doping. For Gram-positive bacteria the antibacterial efficacy changed with silver doping from 71% to 99.9%. It is obvious that the Ag + HA layers, with 4.4%; 8.3% and 13.7% of Ag, exhibited excellent anti-bacterial ability. For Gram-negative bacteria the efficacy changed from 3.9% to 100%. Antibacterial efficacy of 100% was found for silver doped HA layers of 8.3 and 13.7% Ag concentrations. No influence of crystallinity on bacterial efficacy was observed. Amorphous and polycrystalline hydroxyapatite and doped polycrystalline layers were non-toxic. Silver doped amorphous HA layers were mildly toxic from 1.2% Ag. Acknowledgments We would like to thank the grant SGS12/167/OHK4/2T/17 of the Czech Technical University, Prague, grant COST LD 12069, Institutional Research Plan AVOZ50200919 and the grant of the Ministry of Education of the Czech Republic (MSM 6198959216).
References
[1] A.B.G. Lansdown, in: G. Burg (Ed.), Biofunctional Textiles and the Skin, Curr Probl. Dermatol, Basel, Karger, 2006. [2] Y. Chen, X. Zheng, Y. Xie, Ch. Ding, H. Ruan, C. Fan, J. Mater. Sci. Mater. Med. 19 (2008) 3603–3609. [3] W. Chen, Y. Liu, H.S. Courtney, M. Bettenga, C.M. Agrawal, J.D. Bumgardner, J.L. Ong, Biomaterials 27 (2006) 5512–5517. [4] R.J. Chung, M.F. Hsieh, K.C. Huang, L.H. Perng, F.I. Chou, T.S. Chin, J. Sol–Gel Sci. Technol. 33 (2005) 229–239. [5] W. Chen, S. Oh, A.P. Ong, N. Oh, Y. Liu, H.S. Courtney, M. Appleford, J.L. Ong, J. Biomed. Mater. Res. A 82A (2007) 899–906. [6] N. Rameshbabu, T.S.S. Kumar, T.G. Prabhakar, V.S. Sastry, K.V.G.K. Murty, K.P. Rao, J. Biomed. Mater. Res. A 80A (2007) 581–591. [7] Q.L. Feng, F.Z. Cui, T.N. Kim, J.W. Kim, J. Mater. Sci. Lett. 18 (1999) 559–561. [8] K.S. Oh, S.H. Park, J.K. Jeong, Mater. Sci. Forum 449–452 (2004) 1233–1236. [9] G. Gosheger, J. Hardes, H. Ahrens, A. Streitburger, H. Buerger, M. Erren, A. Gunsel, F.H. Kemper, W. Winkemann, C. Eiff, Biomaterials 25 (2004) 5547–5556. [10] C. Chamber, C. Proctor, P. Kabler, J. Am. Water Works Assoc. 54 (1962) 208–216. [11] J.W. Choi, H.M. Cho, E.K. Kwak, T.G. Kwon, H.M. Ryoo, Y.K. Jeong, K.S. Oh, H.I. Shin, Key Eng. Mater. 254–256 (2004) 47–50. [12] A.J. Clarke, in: A. Heffner (Ed.), Handbooch der experimentallen Pharmacologie: Erganzung, Springer, Berlin, 1937. [13] C.F. Koch, S. Johnson, D. Kumar, M. Jelinek, D.B. Chrisey, A. Doraiswamy, C. Jin, R.J. Narayan, I.N. Mihailescu, Mater. Sci. Eng. C 27 (2007) 484–494. [14] R.K. Appleyard, Genetics 39 (1954) 440–452. [15] In: M. Madigan, J. Martinko (Eds.), Brock Biology of Microorganisms, 11th Ed., Prentice Hall, 2005. [16] S.E. Luria, Cold Spring Harb. Symp. Quant. Biol. 18 (1953) 237–244. [17] L. Huang, D.Q. Li, Y.J. Lin, M. Wei, D.G. Evans, X. Duan, J. Inorg. Biochem. 99 (2005) 986–993. [18] M. Jelinek, T. Kocourek, K. Jurek, J. Remsa, J. Miksovsky, M. Weiserova, J. Strnad, T. Luxbacher, Appl. Phys. A 101 (2010) 615–620. [19] M. Jelinek, M. Weiserova, T. Kocourek, K. Jurek, J. Strnad, Laser Phys. 20 (2010) 562–567. [20] S.S. Park, H.J. Lee, I.H. Oh, B.T. Lee, Key Eng. Mater. 277–279 (2005) 113–118. [21] K.L. Barbalace, Element Silver http://environmentalchemistry.com/yogi/periodic/ Ag.html. [22] S. Nath, S. Kalmodia, B. Basu, J. Mater. Sci. Mater. Med. 21 (2010) 1273–1287. [23] M.A. Fanovich, M.S. Castro, J.M.P. Lopéz, Ceram. Int. 25 (1999) 517–522.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Antibacterial, cytotoxicity and physical properties of laser — Silver doped hydroxyapatite layers M. Jelinek a, b,⁎, T. Kocourek a, b, J. Remsa a, b, M. Weiserová c, K. Jurek a, J. Mikšovský a, b, J. Strnad d, A. Galandáková e, J.Ulrichová e a
Institute of Physics ASCR v.v.i., Na Slovance 2, 18221 Prague 8, Czech Republic Czech Technical University in Prague, Faculty of Biomedical Engineering, Sitna 3105, Kladno, Czech Republic Institute of Microbiology ASCR v.v.i., Vídeňská 1083, Prague 4, Czech Republic d Lasak Ltd., Papirenská 25, 1600 Prague 6, Czech Republic e The Faculty of Medicine and Dentistry, Palacký University in Olomouc, Hněvotínská 3, 775 15 Olomouc, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 12 June 2012 Received in revised form 27 September 2012 Accepted 3 December 2012 Available online 9 December 2012 Keywords: Hydroxyapatite Silver Thin films PLD Antibacterial Cytotoxicity
a b s t r a c t Hydroxyapatite layers with silver doping from 0.06 at.% to 14 at.% were prepared by laser deposition. The films' physical properties such as morphology, composition, crystallinity, Young's modulus and microhardness were measured. Films were amorphous or polycrystalline in dependence on deposition temperature (from RT to 600 °C). Antibacterial properties were tested using Escherichia coli and Bacillus subtilis cells. The antibacterial efficacy changed with silver doping from 4% to 100%. Cytotoxicity was studied by a direct contact test. Depending on doping and crystallinity the films were either non-toxic or mildly toxic. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Metallic silver and silver compounds as hydroxyapatite (HA)–silver composites, are widely used in medical devices and health care products [1]. Besides the mechanical properties, antibacterial properties and cytotoxicity are also important for medical applications. Bacterial infections are usually caused by the adherence and colonization of bacteria on coated implants [2]. Silver and silver ions have long been known to have strong inhibitory and bactericidal effects as well as a broad spectrum of anti-microbial activities [2]. Antibacterial tests evaluate anti-bacterial performance of silver against bacteria. Different positive and negative strains were used in testing such as: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus [2], Staphylococcus epidermis [3], Streptococcus mutants [4] and Streptococcus pyogeneses [1]. Cytotoxicity is the capacity of an agent to be toxic to cells. Cytotoxicity test of silver doped hydroxyapatite was evaluated with fibroblast cell [2], human embryonic palatal mesenchymal cells [5], human osteoblast cell line [6], murine macrophages [7], agar overlay cytotoxic assay [8], C-reactive proteins, and neutrophilic leukocytes [9]. Various opinions concerning biocompatibility were presented in the ⁎ Corresponding author at: Institute of Physics ASCR v.v.i., Na Slovance 2, 18221 Prague 8, Czech Republic. Tel.: +420 266052733. E-mail address: [email protected] (M. Jelinek). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.12.018
literature: silver exerts its antimicrobial action at low concentrations (1 ppm) [1], no significant cytotoxicity was observed for low Ag concentration [3,6], silver toxicity is dose dependent [4] and can be toxic [8,10–12]. The conclusions published in the mentioned studies are different and hard to compare. The reason is that the properties of doped materials depend on the dopant concentration, shape of material (bulk, thin layer, nanoparticles) and on the methods and parameters used to introduce silver into the material. It is known that different forms of Ag confer different degrees of bactericidal properties [3]. Minimal level of silver ions necessary in any situation to clear infections is not known yet. Further studies are urgently required [1]. One of the frequently used method for fabrication of HA films is pulsed laser deposition (PLD) [13]. In our contribution we studied silver doped hydroxyapatite layers prepared by PLD. Films of a wide scale of silver doping were created. The physical and antibacterial properties and cytotoxicity are presented and discussed. 2. Materials and methods Thin films of silver, HA and Ag + HA were prepared by PLD using a KrF excimer laser (λ = 248 nm, τ = 20 ns, rep. rate of 10 Hz). The laser beam was focused on a silver, HA or Ag + HA target with energy density of 2 J cm −2. The HA target was pressed from HA powder
M. Jelinek et al. / Materials Science and Engineering C 33 (2013) 1242–1246
supplied by Merck Co. The angle between the target normal and laser radiation was 45°. The target was rotated. Substrate (titanium, fused silica or Si(111)) was in a distance of 4 cm from the target. The deposition conditions to grow HA, Ag+ HA and Ag layers was the same. Substrate was held at room temperature or heated up to 600 °C, to create amorphous or crystalline films. Films were grown in a mixture of water vapor and argon of 40 Pa (water — 22 Pa, Ar — 18 Pa). The layers' thickness was determined by Alpha-step IQ mechanical profilometer (KLA Co.). The composition (WDS — wavelength dependence X-ray spectrometer) was analyzed with EDAX Jeol Supersprobe 733. Jeol was also used for the study of morphology (SEM — scanning electron microscopy) measurements. Young's modulus and microhardness were tested by Hysitron Triboindenter® TI950 device with head NanoScratch transducer SN5-483-194 (Pmax = 10 mN). The testing probe was diamond of Berkovich shape. For indentation, quasistatic loads with partial unloads (to 50% of the load) were used, and the time of each phase (load, dwell and unloading time) was 5 s. Used force was from 1 to 5 mN with 1 mN step. On each sample, 9 points (in 3 × 3 matrix) of indents with distances of tested points 20 μm were arranged. 2.1. In vitro tests The antibacterial properties of Ag + HA films were tested on the Gram-negative laboratory strain E. coli K12 C600 [14] and on the Gram-negative strain B. subtilis 168, commonly found in soil [15]. Cells were grown in Lysogeny Broth (LB) [16] medium and plated on LA plates (LA; LB with addition of 15% of agar). Tested silver doped HA layers were placed into sterile glass chambers and covered with 1 ml of the overnight cultures diluted in the sterile physiological saline solution on the concentration of cells 10 6 CFU/ml. Glass chambers were cultivated in 37 °C and aliquots of 100 μl were withdrawn at the time points and serially diluted up to 10 −6 in physiological saline solution. 100 μl of each diluted sample were spread over an agar plate. The plates were incubated overnight and number of growing colonies was counted. The bactericidal efficacy of different silver doped HA layers was estimated according the formula [17]: bactericidal ef f icacyð% Þ ¼
alive number in ref erence group alive number in experimental group ⋅100: alive number in ref erence group
Cytotoxicity was evaluated in six samples, amorphous or polycrystalline, containing pure hydroxyapatite or silver doped hydroxyapatite (doping 1.2% or 4.4%). Cytotoxicity was evaluated using a biocompatibility direct contact test on the mouse Swiss NIH embryo cell line (NIH 3T3), obtained from European Collection of Cell Cultures (UK). The cells were grown in Dulbecco's modified Eagle's medium supplemented with heat-inactivated bovine serum (10%,v/v), streptomycin(100 U/ml) and penicillin (0.1 mg/ml). Cells were incubated in a humidified atmosphere with 5% (v/v) CO2 at 37 °C.
materials, similar shape titan sample (negative control) and copper sample (positive control) were used. After the incubation period the cultures were analyzed using an inversion microscope (Olympus, Japan). Inhibition of cell growth was quantified by taking the distance between the sample and the area where morphologically no differences were seen (decolonization index, lysis index) in comparison to control cultures. After that, the samples and medium were carefully removed, cells were fixed with methanol (1 min), colored with crystalline violet (2%, m/v in 20% methanol; 5 min), properly washed with water and dried. The decolonization index, lysis index and cell response were determined according to ISO 10993-5:2005 and ISO 7405:2008. Finally, the cell response to a test sample is based on the median decolonization index and lysis index of at least three replicate tests. 3. Results and discussion 3.1. Crystallinity The layers created at low substrate temperature were amorphous and the layers created at temperatures higher than 520 °C were polycrystalline [18,19]. Example of XRD of doped HA film is shown in Fig. 1 (only Ag and HA peaks were observed). 3.2. Composition From WDS measurement it follows that Ca/P ratio of our doped and undoped layers is rather higher than that for stoichiometric composition (1.67), but it is nearly unchanged for all silver doping (from zero doping to 14 at.% of Ag) — see Table 1. The result is different from that of [20], where it was observed that Ca/P decreased with an increasing Ag content. Because the ratio is unchanged, we can conclude that the bioresorbability is unchanged for our doped and undoped layers. We also measured the silver content in droplets: it was slightly lower or higher than that measured in smooth parts of layer. We confirmed that the content of droplets is a mix of silver and HA (there is no pure silver). 3.3. Morphology Layers were smoothly covered with small droplets. The presence of small silver content contributes to smoother layers — see Fig. 2. 3.4. Young's modulus (Y) and hardness (H) Young's modulus (Y) and hardness (H) of our amorphous HA and Ag + HA films are in the range from 45 GPa to 8 GPa, respectively from 0.3 GPa to 0.7 GPa (Fig. 3). There is a small change in Y and H
2.2. Biocompatibility direct contact test The direct contact biocompatibility test is based on the addition of a test sample directly on the top of a sub-confluent cell layer (without agar layer in between). The guidance for the test is in accordance with the international standards compiled as ISO 10993-5:2005 (Tests for cytotoxicity — In Vitro Methods) and ISO 7405:2008 (Dentistry — Evaluation of biocompatibility of medical devices used in dentistry).The cells were seeded in Petri dishes (6 cm diameter; 7 ml culture medium) at a density of 1 × 105 cells/ml and incubated in a humidified atmosphere with 5% (v/v) CO2 at 37 °C for 24 h. Then the culture medium was changed to the serum free one and the test sample controls were applied on the top of the cell layer and incubated for 24 h. As reference
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Fig. 1. XRD spectrum of HAAG4.4A layer (4.4 at.% of Ag).
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Table 1 Structure, WDX composition and bactericidal efficacy of HA and Ag + HA layers on fused silica substrates. Ca/P
Bacterial efficacy [%] Gram positive
Bacterial efficacy [%] Gram negative
0
1.9
–
–
–
1.2
–
–
–
–
–
4.4
–
99.9
Amorph.
21.9
13.6
8.3
1.6
–
100
Amorph.
–
–
–
–
100
Amorph.
–
–
100
–
–
99.2
Polycryst.
–
–
0
–
–
–
Polycryst.
26.1
13.6
0.06
1.9
71.0
3.9
Polycryst.
24.9
14.2
0.3
1.7
71.8
–
Polycryst.
25.8
13.4
1.2
1.9
93.4
99.9
Polycryst.
25.3
12.9
4.4
1.9
99.9
–
Polycryst.
22.2
11.5
13.7
1.9
99.9
100
Sample
HA structure
Ca [at.%]
P [at.%]
Ag [at.%]
HAAG 0.0A HAAG 1.2A HAAG 4.4A HAAG 8.3A HAAG 13.7A HAAG 100A HAAG 0.0C HAAG 0.06C HAAG 0.3C HAAG 1.2C HAAG 4.4C HAAG 13.7C
Amorph.
25.9
13.7
Amorph.
–
Amorph.
13.7
moduli with increase of silver content. For amorphous samples the Y and H moduli are significantly lower than that for crystalline samples. For polycrystalline samples the Y and H moduli are in the range from 141 GPa to 100 GPa, respectively from 6.8 GPa to 3.0 GPa (Fig. 3). In polycrystalline layers there is also a small change in Y modulus with silver concentration. As to hardness we observed a decrease of H with silver increase. The value of Y and H moduli depends on the substrate material (Fig. 3). The measured dependences are partly in agreement with [20] where the decrease of Y and H was observed with an increase of Ag concentration. From literature it follows that the Y and H moduli of pure HA are in the range from 85 GPa to 90 GPa and from 2.4 GPa to 5.9 GPa, respectively [20–23]. The Y and H moduli of silver are 82.7 GPa and 0.15 GPa–1.8 GPa, respectively [19]. We see that our measurements of amorphous layers show lower values and the results reached with polycrystalline layers show higher values than those we can find in the literature.
Fig. 3. Young's modulus (top) and hardness (below) for amorphous and crystalline Ag + HA samples prepared on silicon and titanium substrates.
3.5. In vitro tests HA layers doped with various concentration of silver (0.06%; 0.3%; 1.2%; 4.4%; 8.3% and 13.7%) and various structures were tested for their antimicrobial effect against the E. coli and B. subtilis strains. Data presented are an average of 5 measurements. 3.5.1. Gram-positive strain The number of B. subtilis colonies was reduced both in time and with a silver concentration. Except of layers with very low content of silver (0.06%) all analyzed tablets exhibited a bactericidal efficacy even after 1 h of cultivation with the bacteria. After 24 h of cultivation the bactericidal efficacy increased gradually to 99.9% (for all samples with doping of 4.4% Ag or higher). In summary, for silver
Fig. 2. SEM photo of HA layer (a — thickness of 300 nm), and Ag + HA layers (b — 4 at.% Ag, thickness of 500 nm, c — 13.7 at.% Ag, thickness of 800 nm). Magnification 400 ×. Maximum size of droplets: a) 8 μm, b) 5 μm, and c) 8 μm.
M. Jelinek et al. / Materials Science and Engineering C 33 (2013) 1242–1246 Table 2 Cytotoxicity of HA and doped Ag + HA layers. Sample
HAAG 0.0A HAAG 1.2A HAAG 4.4A HAAG 0.0C HAAG 1.2C HAAG 4.4C
Decolonization index/lysis index
Cytotoxicity Interpretation
0
0/0
0
Non-cytotoxic
0.83
1/1
1
Mildly toxic
Decolonization index [mm] I.
II.
III. Average
0
0
0
1
0.5 1
1.5 1
2
1.5
1/1
1
Mildly toxic
0
0
0
0
0/0
0
Non-cytotoxic
0
0
0
0
0/0
0
Non-cytotoxic
0
0
0
0
0/0
0
Non-cytotoxic
doping in region from 0.06% to 13.7% the antibacterial efficacy changed from 71% to 99.9% (Table 1). All tested layers were polycrystalline.
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3.5.2. Gram-negative strain Doped layers were tested for their antimicrobial effect against the strain E. coli C600. Comparison of their bactericidal efficacy expressed as percentage of killed cells is presented in Table 1. Except of layers with very low content of silver (0.06%Ag) all analyzed tablets exhibited excellent bactericidal efficacy (> 99.9%). Layers with silver concentration higher than 8.3% reached nearly 100% efficacy both for polycrystalline and amorphous layers. Cytotoxicity was evaluated in six samples, amorphous or polycrystalline, containing pure hydroxyapatite or silver doped hydroxyapatite (doping 1.2% or 4.4%). Neither visible alterations to NIH 3T3 monolayer nor cytotoxic effects (destruction of cells, decolonized zone, lysis cells) were found using inversion microscope and crystalline violet incorporation to amorphous and polycrystalline hydroxyapatite (HAAG0.0A and HAAG0.0C) and doped polycrystalline hydroxyapatite (HAAG1.2C and HAAG4.4C) samples. The results indicate that specimens were non-toxic (interpretation decolonization index/index lysis 0/0) and fully biocompatible with the cell model used in the study (Table 2, Fig. 4c–f). Mild cytotoxic effect (mild
Fig. 4. Effect of tested specimens on the NIH 3T3 cell line. a) Titan sample (negative control); b) cooper sample (positive control); c) HAAG0.0A; d) HAAG0.0C; e) HAAG1.2C; f) HAAG4.4C; g) HAAG1.2A and h) HAAG4.4A. The samples were observed at magnification 100 ×.
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destruction of cells, decolonization zone characteristic for lyses cell 1/1) was observed in doped amorphous (HAAG1.2A and HAAG4.4A) samples (Table 2, Fig. 4g and h). Our results of cytotoxicity test partly agree with conclusions in [2] where they found that for plasma sprayed silver doped HA coating the layers containing b5% of silver have no cytotoxic effect. 4. Conclusions The silver doped HA layers with doping of 0.06%, 0.3%, 1.2%, 4.4%, 8.3% and 13.7% were fabricated by PLD method. Depending on deposition conditions the films were amorphous or polycrystalline. We observed a distinct difference in Young's modulus Y and hardness H for amorphous and polycrystalline HA and Ag + HA films and the decrease in Y and H with silver doping. For Gram-positive bacteria the antibacterial efficacy changed with silver doping from 71% to 99.9%. It is obvious that the Ag + HA layers, with 4.4%; 8.3% and 13.7% of Ag, exhibited excellent anti-bacterial ability. For Gram-negative bacteria the efficacy changed from 3.9% to 100%. Antibacterial efficacy of 100% was found for silver doped HA layers of 8.3 and 13.7% Ag concentrations. No influence of crystallinity on bacterial efficacy was observed. Amorphous and polycrystalline hydroxyapatite and doped polycrystalline layers were non-toxic. Silver doped amorphous HA layers were mildly toxic from 1.2% Ag. Acknowledgments We would like to thank the grant SGS12/167/OHK4/2T/17 of the Czech Technical University, Prague, grant COST LD 12069, Institutional Research Plan AVOZ50200919 and the grant of the Ministry of Education of the Czech Republic (MSM 6198959216).
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